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Full text of "Interactions between nematode and fungal pathogens of the citrus fibrous root cortex"

INTERACTIONS BETWEEN NEMATODE AND FUNGAL PATHOGENS 
OF THE CITRUS FIBROUS ROOT CORTEX 




FAHIEM E. EL-BORAI 






A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 



2001 















To The Memory Of My Brother Samir 
My Mother and My family 



ACKNOWLEDGMENTS 

I would especially like to thank to Dr. Larry Duncan, my supervisor, for his 
friendship and guidance throughout my graduate program. He provided me with 
intellectual, thoughtful discussion, encouragement, and time. Without his help and 
support I would not have come from Egypt and continued at the University of Florida. 
His high ethical standards and philosophical views will never be forgotten. I would like 
to thank Dr. James Graham, for all of his support, guidance and friendship. He was very 
kind and helpful whenever I needed his help. I also thank the other members of my 
supervisory committee, Dr. Don Dickson, Dr. R. McSorley and Dr. James Nation for their 
kindness and valuable, thoughtful discussion toward my research. Especially warm 
thanks go to Denise Dunn and Jason Zellers who assisted me any time I needed them. 
Their wonderful friendship and company made it easy for me to do my research in Lake 
Alfred. My appreciation goes to Diana Drouillard and Diane Bright who assisted me in 
the lab. I would like to thank my dear friends Dr. Mukaddes Kayim, Kanjana 
Mahattanatawee, Zenaida Viloria at Lake Alfred. My great thanks go to Kamal and his 
wife Faten and their lovely daughter Yasmin, Ahmad and his wife Azza, for their 
friendship and help. My appreciation to my dear friend Janete Brito for her friendship 
and thoughtful discussion. I would like also to thank my professors back in Egypt, Dr. 
Ahmad A. Salem and Dr. Moustafa E. Mahrous, who taught me what nematology is and 
opened doors giving me a chance to study this wonderful science. My great thanks go to 



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my family, my constant prayers go to my older brother, Samir, who passed away 3 
months after I came to the United States. His dream was to see me graduate from the 
University of Florida, and I will never forget him, God bless him. My lovely wonderful 
mother, brothers and sisters always encouraged me to follow my dream, and without their 
love and support, I could never be who I am today. Finally, my appreciation goes to the 
Egyptian Government for giving me the financial support necessary to obtain my Ph.D 
from the University of Florida. 






IV 



TABLE OF CONTENTS 

Page 

ACKNOWLEDGMENTS iii 

LIST OF TABLES vii 

LIST OF FIGURES viii 

ABSTRACT x 

CHAPTERS 

1 GENERAL INTRODUCTION 1 

2 REVIEW OF LITERATURE 3 

Introduction 3 

Defining Interaction 4 

Tylenchulus semipenetrans 6 

Biology of Tylenchulus semipenetrans 6 

Damage Symptoms 8 

Environmental Factors 8 

Phytophthora nicotianae 12 

Biology of Phytophthora spp 13 

Damage Symptoms 14 

Environmental Factors 14 

Disease Complexes Involving Nematodes and Fungi 15 

Mechanisms of Interactions between Nematodes and Fungi 22 

Disease Complexes Involving Citrus Nematodes and Fungi 30 

Effects of Fungus on Nematode Populations 31 

Nematode and Rhizosphere Microbial Community Structure 32 

3 INFECTION OF CITRUS ROOTS BY TYLENCHULUS SEMIPENETRANS 
REDUCES ROOT INFECTION BY PHYTOPHTHORA NICOTIANAE 38 

Introduction 38 

Materials and Methods 39 

Results 43 

Discussion 45 



4 TYLENCHUL US SEMIPENETRANS ALTERS THE MICROBIAL COMMUNITY IN 
THE CITRUS RHIZOSPHERE 54 

Introduction 54 

Materials and Methods 56 

Results 61 

Discussion 67 

5 EGGS OF TYLENCHULUS SEMIPENETRANS INHIBIT GROWTH OF 
PHYTOPHTHORA NICOTIANAE AND FUSARIUM SOLANI IN VITRO 81 

Introduction 81 

Materials and Methods 82 

Results 85 

Discussion 87 

6 RESEARCH SUMMARY AND CONCLUSION 96 

LITERATURE CITED 101 

BIOGRAPHICAL SKETCH 119 












VI 



LIST OF TABLES 

Table Page 

3-1. Effect of soil pH (low = 4.5 and high = 7.0) on final population density 

of the citrus nematode, Tylenculus semipenetrans 52 

3-2. Analyses of variance of effects of Tylenchulus semipenetrans on stem fresh 
weight, root fresh weight and Phytophthora nicotianae protein in citrus roots 
in the laboratory 52 

4-1. Total of bacteria colony forming units isolated from root segments 

infected and uninfected by Tylenchulus semipenetrans collected from two different 
locations 72 

4-2. Numbers of colony forming units of Bacillus megaterium, Burkholderia cepacia, 
and Fusarium solani isolated from root segments infected and uninfected by 
Tylenchulus semipenetrans collected from three different locations 73 

4-3. Analyses of variance of effects of Burkholderia cepacia, Bacillus megaterium, and 
Tylenchulus semipenetrans on stem fresh weight, root fresh weight and 
Phytophthora nicotianae protein in citrus roots in the laboratory 77 

4-4. Analyses of variance of effects of Burkholderia cepacia, Bacillus megaterium, and 
Tylenchulus semipenetrans on stem fresh weight, root fresh weight and 
Phytophthora nicotianae protein in citrus roots in the greenhouse 78 

5-1 . Effect of cupric sulfate, mercuric chloride and streptomycin sulfate on Phytophthora 
nicotianae mycelial growth after 48 hours in vitro 95 



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LIST OF FIGURES 

Figure Page 

3-1 Phytophthora nicotianae infection of Tylenchulus semipenetrans infected and 

uninfected citrus root segments in vitro 50 

3-2 Effect of Tylenchulus semipenetrans on growth and pathogencity of 

Phytophthora nicotianae 51 

3-3 Effect of pH and the citrus nematode Tylenchulus semipenetrans on growth in 

roots of Phytophthora nicotianae and damage by the fungus to citrus seedlings 53 

4-1 Effect of nematode-associated bacteria on Phytophthora nicotianae mycelial 

growth in vitro 74 

4-2 Effect of Burkholderia cepacia, Bacillus megaterium, Tylenchulus semipenetrans 
and Phytophthora nicotianae on citrus seedlings root fresh weight in the laboratory 
and greenhouse 75 

4-3 Effect of Burkholderia cepacia, Bacillus megaterium, Tylenchulus semipenetrans 
and Phytophthora nicotianae on citrus seedlings stem fresh weight in the laboratory 
and greenhouse 76 

4-4 Average numbers of bacteria colony forming units recovered from citrus seedlings 

in the laboratory and greenhouse 79 

4-5 Effect of Burkholderia cepacia, Bacillus megaterium, and Tylenchulus 

semipenetrans on absorbance of Phytophthora nicotianae protein in citrus 

roots (measured by ELISA test)in the laboratory and greenhouse 80 

5-1 Effect of Tylenchulus semipenetrans eggs (extracted with and without bleach) on 

Phytophthora nicotianae mycelial growth after 48 hours in vitro 90 

5-2 Effect of Tylenchulus semipenetrans eggs on Phytophthora nicotianae 

and Fusarium solani mycelial growth after 48 hr in vitro 91 

5-3 Effect of Tylenchulus semipenetrans eggs (extracted with and without bleach) on 
Fusarium solani mycelial growth after 48 hours (experiment A, B and 36, 72 in 
experiment C) in vitro 92 



vin 



5-4 Effect of Meloidogyne arenaria eggs (extracted with and without bleach) on 
Phytophthora nicotianae (A) and Fusarium solani (B) mycelial growth 
after 48 hours in vitro 93 

5-5 Effect of Meloidogyne arenaria eggs on Phytophthora nicotianae (A) and 

Fusarium solani (B) mycelial growth after 48 hours in vitro 94 












IX 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Doctor of Philosophy 



INTERACTIONS BETWEEN NEMATODE AND FUNGAL PATHOGENS 
OF THE CITRUS FIBROUS ROOT CORTEX 

By 

Fahiem E. El-Borai 
December 2001 

Chairman: Larry W. Duncan 

Major Department: Entomology and Nematology 

The most commonly encountered association between nematodes and fungi in 
citrus occurs between the citrus nematode Tylenchulus semipentrans Cobb and the root 
rot fungus Phytophthora nicotianae(=parasitica) Dastur Breda de Harm. The fibrous 
root system of citrus trees in Florida and worldwide is commonly infected by both 
parasites. The efficacy of nematicides to manage T. semipenetrans in field experiments 
was directly related to the population density of P. nicotianae, implying significant 
competition between these two parasites. We initiated a study of this hypothesis by 
testing whether infection of root segments by the citrus nematode impeded root infection 
by P. nicotianae in vitro, and by determining the effect of the nematode on growth and 
pathogenicity of the fungus in whole plant experiments. Plants were infected by the 



fungus, the nematode, both organisms or neither organism. Infection of roots by T. 
semipenetrans reduced subsequent infection by P. nicotianae and damage to the plant. 
Citrus seedlings infected by both organisms grew larger and contained less fungal protein 
in the root tissues than did plants infected by only the fungus thus demonstrating 
antagonism of the nematode to the fungus. 

Two hypotheses were investigated to explain the mechanism of the interaction 
between these pathogens: 1) indirect mediation through increased colonization of 
nematode feeding sites by microorganisms antagonistic to P. nicotianae, and 2) direct 
antibiosis by the nematode. To test the first hypothesis, a field survey was initiated to 
determine whether infection by T. semipenetrans changes the composition of rhizosphere 
inhabiting microorganisms, and to identify microorganisms that are consistently 
associated with the nematode. Results showed that T. semipenetrans altered the 
microbial community in the citrus rhizosphere by increasing propagule densities of 
bacteria and fungi. The dominant bacterial species isolated were Bacillus megaterium 
and Burkholderia cepacia. Both bacteria were used in whole plant factorial experiments 
with P. nicotianae and T. semipenetrans either alone or in combination. Neither 
bacterium inhibited growth of the fungus when inoculated alone. Nevertheless, the 
nematode and both bacteria increased the growth of citrus seedlings infected by P. 
nicotianae. 

To test the second hypothesis, in vitro bioassays were conducted to determine the 
effects of eggs of two nematodes T. semipenetrans and Meloidogyne arenaria on 
mycelial growth of P. nicotianae and Fusarium solani. Tylenchulus semipenetrans eggs 
suppressed mycelial growth of P. nicotianae and F. solani in vitro, but M. arenaria eggs 



XI 



had no comparable effect on either fungus. This research showed an antagonistic effect 
of a plant parasitic nematode on a plant pathogenic fungus and shows potential 
mechanisms involving direct inhibition of the fungus by the nematode and indirect 
mitigation of fungal virulence mediated by complex microbial interactions in the citrus 
rhizosphere. 









Xll 



CHAPTER 1 
GENERAL INTRODUCTION 

Citrus is the most economically important crop in Florida with revenues 
exceeding one billion dollars every year. Approximately 336,817 ha of citrus are grown 
in Florida. Of these, 80% are orange, 14% are grapefruit, and 6% are specialty fruit 
(Florida Agriculture Statistics Service 2000). The most important citrus-growing regions 
in Florida are located in Polk, Hendry, Highlands, DeSoto, Hardee, St. Lucie, Indian 
River, and Martin counties. These eight counties comprised about 75% of the total area 
of citrus production in Florida (Florida Agriculture Statistics Service 2000). 

More than 100 biotic and abiotic factors cause diseases of citrus trees (Whiteside 
et al. 1988). Two of the most important biotic factors for young and mature citrus trees 
are fungi and nematodes. Diseases caused by soilborne fungi, such as Phytophthora spp. 
result in root rot; foot rot; brown rot of fruit; reduced fruit quality and yield; and under 
optimum conditions for disease trees may be killed (Timmer et al. 1989). Citrus 
nematode Tylenchulus semipenetrans (Cobb 1914) is ubiquitous in commercial citrus 
growing regions worldwide. The nematode is the causal agent of the disease citrus slow 
decline. The fibrous root systems of citrus trees are commonly infected by both T. 
semipenetrans and Phytophthora nicotianae Dastur Breda de Haan (synonym = P. 
parasitica) (Hall 1993). Both organisms feed in the cortex of fibrous roots and both have 
been shown to reduce the density of the fibrous root system (Duncan et al. 1993; Graham 
and Menge 1999). In a study of the effects of managing concomitant populations of 

1 



Phytophthora nicotianae and Tylenchulus semipenetrans, Graham and Duncan (1997) 
showed that yields responded in a density-dependent manner to the control of either or 
both parasites. However, management of both pathogens did not substantially increase 
yields more than management of either alone. Reduction of either population increased 
the population density of the other. Increased root growth after fungus control is 
responsible for increased nematodes, because the numbers of nematodes per mass of root 
did not increase. Root mass did not increase following nematicide treatments, whereas 
there was a highly significant inverse relationship between numbers of nematodes and 
numbers of propagules of P. nicotianae in the soil. This suggests that either the 
nematode or an associated agent was producing antibiotics affecting the fungus, or that 
the nematode competed for resources with the fungus. These results were supported by a 
greenhouse study in which T. semipenetrans interfered with P. nicotianae, reducing 
levels of infection in roots and producing increased growth of citrus seedlings compared 
with seedlings infected by P. nicotianae alone. The research revealed a significant 
interaction between the nematode and the fungus that may be of economic importance. 
The objectives of this research were to extend the findings of Graham and Duncan (1997) 
by: 

• determining the nature of the association (additive, or antagonistic or synergistic) 
between citrus nematode Tylenchulus semipenetrans and root rot fungus 
Phytophthora nicotianae on citrus plants. 

• determining the mechanism(s) underlying the interaction. 






CHAPTER 2 
REVIEW OF LITERATURE 

Introduction 

Under natural conditions a plant is a potential host to various microorganisms that 
can influence each other, particularly when they occupy the same niche. It is reasonable 
to expect that infection by one pathogen may alter the host physiology and thereby 
subsequent infection by another organism (Taylor, 1990). Pathogens also affect one 
another through resource competition for nutrients (Elad and Chet, 1987, Suslow, 1982, 
Weller 1985), competition for infection sites, (Baker, 1968, Osburn et al., 1989) or 
parasitism and production of lytic enzymes (Mitchell and Alexander, 1961; Mitchell and 
Hurwitz, 1965; Sneh, 1981). Weller (1988) reported that nutrients, rather than space, are 
thought to be the limiting factor in competition among rhizosphere microorganisms 
during colonization. Plant-parasitic nematodes often play a major role in disease 
interactions involving a variety of other organisms. Interactions involving nematodes are 
important because they contribute substantially to variability in crop growth (Zadoks and 
Schein, 1979). The most commonly cited interactions involving nematodes include the 
following: 

• Synergistic disease complexes (Powell, 1971a, 1971b, 1979; Golden and Van Gundy, 
1975). 

• Antagonism, or disease inhibition (Jorgenson, 1970, Gray et al., 1990). 

• Reduced resistance to subsequent infection (Harrison and Young, 1 941 ; Webster, 



1985; Hasan, 1985; Khan and Nejad-Hosseini, 1991). 
• Increased resistance against a subsequent parasite (Sidhu and Webster, 1981). 

Defining Interaction 

The term interaction is used widely in plant nematology for various kinds of 
associations and its precise usage is rather rare. Both quantitative and qualitative 
responses resulting from two or more factors involved in plant diseases have been 
described as interaction in nematology literature. Nematodes like all organisms come 
into association with other organisms in the course of their existence. Without any 
consideration whether or not "interaction" (in the mathematical sense) operates, 
biologists are prone to see "interaction" when terms like "association," "relationship" or 
just "relation" suffice (Burrows, 1987). Wallace (1983) suggested that use of the term 
"interaction" should be restricted to quantitative plant disease interactions showing 
synergism or antagonism. This would be interaction in the statistical sense rather than in 
the descriptive sense. According to Burrows (1987), all nonstatistical qualitative 
interactions can be simply termed "relationship" or "relation." 

Similarly, use of the term "synergism" and "antagonism" has been variable, with 
different connotations. Dickinson (1979) described how the word synergism was 
introduced in plant pathology and was defined as "an association of two more organisms 
acting at one time and affecting a change that only one is not able to make." Powell 
(1979) defined synergism as "the concurrent or sequential pathogenesis of host plant by 
two or more pathogens in which the combined effects of the two pathogens are greater 
than the sum of the effects of each pathogen alone." The first definition is somewhat 
more qualitative whereas the definition of Powell (1979) is quantitative and more useful 
in nematology. In statistics, the term synergism is used to indicate a positive interaction ( 



i.e. the sum of the treatment effects is not simply additive) (Wallace, 1983, 1989). 
Wallace (1983) proposed the avoidance of the terms synergism and antagonism 
altogether and suggested that the events could be adequately described by the terms 
positive and negative interactions, respectively. 

The statistical definitions of additivity and interaction (antagonistic or synergistic) 
are used in this dissertation. It should be noted, however, that the statistical definition of 
additivity is somewhat at variance with normal usage of the word in a biological sense 
(Duncan and Ferris, 1982). These differences are shown if we consider the plant or 
nematode response to increasing population densities of a single nematode species. A 
typical damage function describing yield as a function of initial nematode population 
density (Pi) is sigmoid. The relationship can be linearized by log transformation of Pi. 
Thus, each successive increment of yield loss is associated with increasingly larger 
cohorts of pathogens. These functions illustrate that each successive infection by a 
nematode causes proportionately less yield loss than did the preceding nematodes, and 
they represent an additive effect. The same phenomenon occurs with respect to 
population growth. Because of intraspecific competition for resources, population 
growth rate is inversely proportionate to Pi. The biological and mathematical basis for 
this phenomenon was described by Nicholson (1933) and extended to nematology by 
Seinhorst (1965). Seinhorst's model was extended to describe more than a single 
nematode species (Duncan and Ferris, 1982). In practical terms, additive effects on yield 
or population growth involving one or several species can be predicted by these models. 
Therefore, any system involving more plant damage or population growth than predicted 
by these models could be considered synergistic, while any involving less damage or 



population growth could be considered antagonistic. Any system predicted by the 
models would be additive; that is, no interaction occurred. 

Tylenchulus semipenetrans 

Tylenchulus semipenetrans was observed for the first time in 1912 by J. R. 
Hodges, a horticultural inspector for Los Angeles County, California (Thomas, 1913). 
Subsequent reports showed that this nematode is widespread and occurs in all citrus- 
producing regions of the world (Heald and O'Bannon, 1987; Van Gundy and Meagher, 
1977). The citrus nematode is the causal agent of a disease called citrus slow decline. 
This disease is so named because the nematode population increases slowly throughout 
the root system and requires several years to debilitate trees and reduce fruit yield (Cohn 
et al., 1965; Reynolds and O'Bannon, 1963). Symptoms of the disease appear sooner 
when trees are replanted in infested soil. High population densities of T. semipenetrans 
in replanted soil was reported to kill young trees within the first year of replanting 
(Thome, 1961). 
Biology of Tylenchulus semipenetrans 

The life stages of T. semipenetrans consist of the egg stage, four juvenile stages 
(J1-J4) and the adult stage (Cobb, 1914; Van Gundy, 1958). The life cycle from egg to 
egg is completed within 6-8 weeks, depending on the host, average soil temperature and 
other environmental factors (Cohn, 1965; O'Bannon et al., 1966; Van Gundy, 1958). 
Development of the first-stage juvenile proceeds within the egg where the first molt 
occurs (Gutierrez, 1947). All stages parasitize root parts except eggs, Jl and males (Van 
Gundy, 1958). The J2, J3, and J4 stages feed on young citrus feeder roots (epidermal and 
hypodermal cells (Cohn, 1965; Schneider and Baines, 1964; Van Gundy and Kirkpatrick, 
1964). Juveniles take approximately 2 weeks to become associated with the roots and 



begin feeding on the epidermal cells (Van Gundy, 1958). Penetration occurs 19 days 
after inoculation on average. Then immature females (J4) complete the infection phase 
within 5 to 6 days (Conn, 1964) by penetrating deeply into feeder root cortical tissue. 
The females increase in size becoming posteriorly swollen and saccate. The mature 
female is a sessile semi-endoparasite. The anterior portion of the female extends several 
cell layers deep in the cortical parenchyma, whereas the posterior portion of the body 
enlarges outside the root. The nematode begins to lay eggs, that remain attached to the 
body in a gelatinous matrix on the root surface (Van Gundy, 1958). The eggs and the 
gelatinous matrix together with its contents are known as eggmasses (Maggenti, 1962). 
Egg masses contain up to 75 to 100 eggs (Baines, 1950). Reproduction is facultatively 
parthenogenic and the mature female lays about 500 eggs during her lifetime (Van 
Gundy, 1958). 

Females feed in the fibrous root cortex where they became immobile establishing 
permanent specialized feeding sites. The feeding site consists of 6 to 10 "nurse" cells 
around the nematode head (Van Gundy, 1958). The "nurse" cells are required for 
successful reproduction and die when the female dies. Nurse cell histology shows the 
cytoplasm to be dense, but devoid of starch, presumably a primary nutrient of the 
nematode (Conn, 1965; Duncan et al, 1994). Root penetration may extend to the 
endodermis (Cohn, 1964; Van Gundy, 1958) and damage is usually pronounced in the 
cortex. Heavily infected roots show extensive necrosis which gives them an abnormally 
dark color compared to noninfected roots. The cortex of the affected region sloughs off, 
resulting in a shortened, irregular appearance or death of the affected rootlet (Cohn, 1965; 
Reynolds and O'Bannon, 1963). 



8 



Damage Symptoms 

Symptoms of slow decline disease vary and are associated with root disfunction. 
Reduced root terminal growth was the first reported clinical symptom (Thomas, 1913). 
At high population density (4,000 females/gram of root), trees generally exhibit low 
vigor, chlorosis, canopy thinning, twig dieback, smaller than normal fruit and reduced 
yield (Cohn et al., 1965). Symptoms of slow decline vary with soil environment. In 
California high population densities of nematodes are invariably associated with reduced 
fruit yield but not always associated with decline symptoms in the tree (Baines et al., 
1978). Population densities of the nematodes in Florida and other parts of the humid 
tropics and subtropics are generally lower than those from dryland or Mediterranean 
climates (Duncan and El Morshedy, 1996). O'Bannon (1968) found that in well-drained 
deep sandy soils in the central ridge of Florida, the population densities of T. 
semipenetrans on mature trees may exceed 5,000 juveniles/g fresh roots, without 
showing distinct decline or visual symptoms. On the other hand, population densities of 
T. semipenetrans below 1000 juveniles/g roots were related to severe decline and more 
obvious symptoms in the poorly drained soil in the coastal areas of Florida where organic 
matter is high; soil is more shallow and the water table and salinity are higher. 
Environmental Factors 

Many biotic and abiotic factors have been shown to influence population densities 
of T. semipenetrans. Gutierrez (1947) was the first to report that temperature greatly 
influenced development of the citrus nematode T. semipenetrans. Only slight nematode 
infection of orange roots occurred at 15 or 35 °C. The optimum nematode infection and 
development was observed between 25 and 30 °C (Baines, 1950). Van Gundy (1958) 






showed that at 25 °C the life cycle from egg to egg required 6-8 weeks. O'Bannon et al. 
(1966) showed that the optimum average temperature for T. semipenetrans population 
development is 25 °C and the percentage of infection was reduced and reproduction was 
delayed at 30 °C. At 25 °C, 80% of penetration occurred when the roots were in the 
primary stage of development and before the appearance of secondary xylem (Conn, 
1964). Van Gundy (1984) showed that the juveniles are not active when mean soil 
temperature is below 16 °C. Van Gundy and Tsao (1963) reported that T. semipenetrans 
reproduction begins at 21 to 22 °C, reached a maximum between 28 and 31 °C and 
ceased at 31 °C. 

In Florida T. semipenetrans females have the highest rate of development in 
summer through autumn (July-Nov.) but development declines during winter (Dec- 
March) and reaches the lowest levels during midsummer (O'Bannon et al., 1972; Duncan 
and Conn, 1990). O'Bannon (1968) showed that fall populations are generally higher 
than spring populations because the reduction in nematode numbers is not as great during 
the summer months. From January to March and July to September conditions are not 
favorable for citrus nematode activity (O'Bannon, 1968). 

Van Gundy and Martin (1961) found higher T. semipenetrans population densities 
in alkaline than in acid soils. The optimum soil pH for T. semipenetrans development is 
6.0-7.5 (Van Gundy et al., 1964), but infection occurs at low soil pH (Bello et al., 1986; 
Davide, 1971; Martin and Van Gundy, 1963; Reynolds et al., 1970). 

Soil with a high organic matter content can greatly influence and favor the initial 
rate of nematode infection and subsequent reproduction. Van Gundy et al. (1964) found 
that nematode infected citrus seedlings grown in soil containing 10-15% clay had the 



10 

highest rate of nematode reproduction and the greatest plant growth reduction. Van 
Gundy (1958) and O'Bannon et al. (1966) suggested that peat moss created a thin 
protective cover layer over infected citrus roots that enhanced nematode infectivity and 
that T. semipenetrans females accumulate in the greatest numbers by adding peat moss to 
soil. 

The production and development of eggs of citrus nematode were reduced in soils 
with low oxygen availability (Stolzy et al., 1963). Population development of T. 
semipenetrans was favored by dry rather than wet soil (Van Gundy et al., 1964). The 
reduction of soil oxygen due to excess soil moisture generally reduces T. semipenetrans 
population densities (Van Gundy et al., 1964; Norton, 1978). Ayoub (1980) found that 
the population densities of T. semipenetrans increased when heavy rains were interrupted 
by short drought spells suggesting that juveniles and eggs were possibly washed out of 
the gelatinous matrix by rain. Soil moisture has a major effect on the seasonal variation 
in population density of T. semipenetrans in sandy soil in Florida citrus orchards 
compared to other climatic factors. Duncan et al. (1993) showed that seasonal patterns of 
root quality and annual differences in root abundance and quality were related to 
populations of both the T. semipenetrans and P. nicotianae. Patterns of change in root 
mass density and concentration of root lignin and nonstructural carbohydrate suggested 
annual as well as seasonal variation in the age structure and nutritional value of the 
fibrous root system. Numbers of nematodes were related inversely (P < 0.01) to soil 
moisture and root lignin content, and positively related to starch concentration. The 
population density of T. semipenetrans tends to be higher in drier climates (Cohn, 1966) 
than in humid tropics and subtropics (Duncan, et al. 1993, O'Bannon, et al. 1972). In 



11 

drier climates (Mediterranean and Florida central ridge) where root systems are 
unrestricted by high water tables, citrus produces a deep root system that maintains high 
levels of T. semipenetrans when water is available from other portions of the root system. 
In humid tropics and subtropics (Florida coastal area) where root systems are restricted 
due to high water tables, the density of T. semipenetrans may be regulated by high soil 
moisture during the rainy season and by complete drought during the dry season. The 
major factor in the population differences in these regions is suggested to be moisture 
availability in surface soils (Duncan and El-Morshedy, 1996). In a greenhouse study 
(Duncan and El-Morshedy, 1996) in which citrus seedlings were grown in vertical tubes 
with upper and lower sections, the population development of T. semipenetrans 
responded differently to dry sandy soils depending on whether all or part of the root 
system experienced drought. Under local drought (only lower section was irrigated), 
number of eggs, juveniles and males in the soils and per gram of roots were higher than 
those under nondrought or uniform drought (both upper and lower section were irrigated 
or neither sections was irrigated) respectively. There was no survival for the nematodes 
under uniform drought in soil. They also suggested that the hydraulic lift through the 
root xylem may prolong the activity of the nematodes in dry soils and other rhizosphere 
organisms. 

The interaction between salinity and T. semipenetrans has a major impact on 
citrus. More citrus nematodes and other nematodes were also found around citrus roots 
subjected to continuous high, though tolerable salinity, than around citrus roots grown at 
lower salinity levels (Machmer, 1958). High T. semipenetrans population densities were 
recovered from mature trees irrigated with various soluble salts compared with lower 



12 

population densities from trees irrigated with surface water. Cohn (1976) reported that in 
South Africa, the highest population densities of T. semipenetrans (10,000-40,000 
juveniles/g fresh root) are commonly associated with saline conditions whereas the 
lowest population densities (100-500 juveniles/g fresh root) were associated with 
nonsaline conditions. In Israel, the highest population densities of T. semipenetrans 
occur in the more saline coastal or desert regions (Cohn et al., 1965; Heller et al., 1973). 
Mashela et al. (1992) studied the interaction between T. semipenetrans and salinity 
showing that salinity increases population densities of T. semipenetrans. These salinity 
effects were most likely systemic because when nematodes and salinity were separated in 
seedlings with splitroots, nematode densities were higher than when nematodes were 
alone. Dunn et al. (1998) studied this relationship and showed de novo arginine 
biosynthesis as a response of citrus to salinity stress, concomitant with enhanced 
susceptibility to attack by T. semipenetrans. The effects of salinity stress on nematode 
behavior, arginine content and phenylalanine ammonia lyase (PAL) activity (PAL is a 
key phenolic chemical defense pathway enzyme) suggested that under stress, citrus grows 
more slowly and produces arginine in response to high levels of in vivo ammonia, 
resulting in lower PAL activity and decreased chemical defense against the nematode. 

Phytophthora nicotianae 

Phytophthora spp. are the causal agents of many devastating diseases, the most 
notable resulting in the famous Irish potato famine in Ireland 1845, caused by P. infestans 
(Klinkowski, 1970). Phytophthora spp. attack more than 2000 plant species worldwide, 
including Citrus spp. and other members of the Rutaceae family (Timmer and Menge, 
1988). The most widespread and important Phytophthora spp. that attack citrus are P. 






13 

nicotianae and P. citrophthora. Phytophthora nicotianae Dastur Breda de Haan 
(synonym = P. parasitica) (Hall, 1993) is common and widespread in most citrus 
growing areas worldwide and causes foot rot, gummosis and root rot (Graham, 1990). 
Fibrous root rot is a common problem in citrus nurseries (Sandler et al., 1989) and about 
90% of field nurseries in Florida are infested with P. nicotianae (Fisher, 1993). 
Pytophthora nicotianae does not live freely in the soil (Tsao, 1969) but obtains its 
nutrients from plant tissue (Lutz and Menge, 1986). 
Biology of Phytophthora spp. 

Phytophthora nicotianae can survive in soil or root debris as chlamydospores or 
oospores (Tsao, 1969). Under unfavorable conditions (cool temperature, soil poorly 
aerated, elevated carbon dioxide) chlamydospores are produced (Tsao, 1971) and can 
survive and persist in adverse conditions for a long period of time (Lutz and Menge, 
1986; Malajczuk, 1983). When favorable conditions return, chlamydospores germinate 
directly to produce mycelia or indirectly to produce sporangia and zoospores (Mircetich 
and Zentmeyer, 1970). 

Oospores occur when the Al and A2 matings of the fungus are present. Oospores 
are produced in lower numbers than chlamydospores. Oospores have thick walls and are 
resistant to drying and cold temperature (Lutz and Menge, 1986). They require longer to 
mature than chlamydospores (Ribeiro, 1983) and remain dormant for extended periods of 
time (Malajczuk, 1983). 









Sporangia are the primary reproductive structures and form during normal 
conditions of oxygen and carbon dioxide (Mitchell and Zentmeyer, 1971). Well-aerated 
moist soil is optimal for both production and germination of sporangia (Sommers et al., 
1970). Sporangia may either germinate and form mycelium or release motile zoospores 



14 

(MacDonald and Duniway, 1978). Each sporangium releases from 5 to 40 zoospores, 
which can swim or be carried by moving water or roots. Zoospores are probably 
attracted to the zone of elongation of new roots by nutrients that are naturally excreted 
from this root zone (Morris and Ward, 1992). Once zoospores are in contact with the 
root, they encyst usually in the presence of high concentrations of amino acids (Khew 
and Zentmeyer, 1973). Zoospores can move over long distances between trees by water 
movement from rainfall or irrigation. 
Damage Symptoms 

Fibrous root rot disease is characterized by decay in the fibrous root cortex. The 
cortex becomes discolored and appears water soaked and then turns soft. The fibrous 
roots slough their cortex, leaving only the vascular tissue (the white, thread-like stele) 
(Whiteside et al., 1988). Root rot is more severe in susceptible rootstocks in infested 
nursery soil (Graham, 1995). Young fibrous roots of citrus are much more susceptible to 
root rot than older roots. The young trees may die due to the loss of a significant 
numbers of roots. In older trees, the trees decline, show low vigor, have smaller fruit size 
than normal, and have reduced yields (Graham and Timmer, 1992). 
Environmental Factors 

The minimum temperature for Phytophthora spp. growth varies from 5 to 7 °C 
with different isolates. The optimum temperature for P. nicotianae is 27 to 32 °C and the 
maximum is 37 °C. Hall (1993) reported that most isolates stopped growth at 5 and 
35 °C and about half of the cultures were killed at 40 °C. Duncan et al. (1993) showed 
that in subtropical climates such as in south Florida, seasonal fluctuations in the 
population density are not consistent, although an overwinter decline does occur. 



15 

Numbers of fungal propagules in the soil and the amount of fungal protein in roots (as 
measured by ELISA) were directly related to concentration of ketone sugars in roots and 
to soil temperature and H 2 0. Phytophthora nicotianae is most active during the warmer 
season. Chlamydospores and oospores are formed for survival during unfavorable 
periods. The release of zoospores is optimal in saturated soils (Mircetich and Zentmeyer, 
1970). Root exudates released by living roots attract zoospores (Morris and Ward, 1992). 
Host susceptibility is increased when roots are stressed or damaged (Graham and Menge, 
1999). Optimum condition for infection growth of P. nicotianae are present in soils with 
drainage restricted by hardpans or clay layers or those with shallow water tables that rise 
into the root zone (Graham and Menge, 1999). Frequency and duration of irrigation 
influence the activity off. nicotianae and the predisposition of roots to rot. Excessive 
soil moisture can result in prolonged oxygen deprivation and makes roots more attractive 
to zoospores, increasing infection. In cool weather, populations of P. nicotianae decline 
in Mediterranean climates when the temperature is below 59°F, but in Florida the 
populations do not drop significantly because soil temperature rarely drops below 59°F 
(Graham and Menge, 1999). 

Disease Complexes Involving Nematodes and Fungi 

Various aspects of nematode-fungal interactions are well reviewed (Powell, 
1971a; Bergeson, 1972; Webster, 1985; Mai & Abawi, 1987; Rowe et al., 1987; Evans & 
Haydock, 1993; France & Wheeler, 1993). The best known of these interactions occur 
between root-knot nematodes and Fusarium wilt fungi; however, root knot nematodes 
have long been known for their ability to predispose plants to infection by other 
secondary pathogens. The first published account of biopredisposition was by Atknison 



16 

(1892), who noted a very close relationship between Meloidogyne sp. and Fusarium wilt 
fungi in cotton. The combinations between these pathogens always contribute to 
synergistic interactions and more severe losses from wilt. Many studies have shown 
interactions among all major groups of parasitic fungi and a diversity of nematodes. In 
most interactions involving nematodes, the nematode plays the primary role modifying 
the host (Pitcher, 1978; Powell, 1979; Patel et al., 2000). With some exceptions, 
nematode infections make the host more susceptible or suitable to other pathogens. Some 
fungal pathogens have also been shown to render the host susceptible to the nematode 
(Hasan, 1985). Examples of synergism, antagonism or additive effects between 
nematode and fungi are described below. 

Sedentary Endoparasitic Nematode: Nematodes enter plant tissues completely 
(sessile entirely within roots) or with a large portion of their body (sessile partly within 
roots). Root-knot {Meloidogyne spp.) and cyst nematodes {Heterodera spp.) are often 
primary pathogens with the ability to predispose plants to heavier infection by other 
pathogens such as Fusarium spp., Phytophothora spp. and Rhizocotonia spp. 
Predisposing effects of Meloidogyne spp. were also noted in parasitism by secondary 
fungal pathogens in tobacco, such as Curvularia, Botrytis, Penicilium, and Aspergillis, 
that do not infect plants in the absence of predisposing factors (Powel et al., 1971b). In 
addition to those well-known associations, various systems involving root-knot, cyst and 
reniform nematodes show the three types of associations/interactions that can occur 
between fungi and nematodes. 

Synergism: Disease complexes in which Heterodera spp are primary pathogens 
are well documented. Other pathogens involved include fungi in the genera Fusarium, 



M 



Phytophthora, Pythium, and Rhizoctonia (Powell, 1971a). Whitney (1974) showed that 
the effect on damping off of sugarbeet of Pythium ultimum and Heterodera schachtii in 
combination was synergistic. Roy et al. (1989) found similar synergistic effects in 
pathogenicity tests of two isolates of Fusarium from soybean plants with symptoms of 
sudden death syndrome (SDS). McLean and Lawrence (1993a) confirmed those findings 
and, using a split root system, showing that the influence of the nematode on SDS is 
localized rather than systemic. Rai and Singh (1996) reported that the wilt incidence and 
reduction in plant growth of pigeonpea increased when plants were inoculated with 
Heterodera cajani and F. udum in combination. The histopathological studies revealed 
that the main site of entry of/ 7 , udum is through the injury created by protruding females 
of//, cajani. 

Walker et al. (1999) studied the relationship between root-knot nematode 
Meloidogyne incognita and the fungus Thielaviopsis basicola on cotton. Extensive 
vascular necrosis and sporulation within vascular tissue was observed in plants infected 
by both pathogens compared to plants grown in soil infested with T. basicola alone, 
which showed no evidence of vascular colonization by the fungus. They concluded that 
M. incognita greatly increases the access of T. basicola to vascular tissue. Jonathan and 
Rajendran (1998) found a synergistic interaction between M. incognita and F. oxysporum 
on banana (cv. Rasthali) both in concomitant and sequential inoculations, resulting in 
significant reduction in plant growth. The Panama wilt disease, in terms of corm rot, was 
significantly higher when nematode inoculation followed the fungus and in concomitant 
inoculations of the pathogens. 

Synergistic interactions also occured between M.javanica and F. oxysporum f.sp. 



18 

ciceris on different chickpea cultivars both in concomitant and sequential inoculations 
where resistance of Pusa-212 to the fungus alone is broken in the presence of the 
nematode (Khan and Hosseini, 1991). 

Predisposition of roots by Meloidogyne spp. for damage by different fungi have 
been reported on many crops. On tomato, inoculation of M. incognita 3 weeks before R. 
solani significantly reduced the tomato shoot weight and length compared with 
inoculation of R. solani 3 weeks before M. incoginta (Ferraz and Lear 1976; Singh et al., 
1981). M. incognita and Fusarium oxysporum f. sp. lycopersici, acting alone caused 
characteristic root galling and shoot wilting, respectively, and significantly reduced plant 
growth and yield. In concomitant inoculation, severity of fusarial wilt was significantly 
increased and plant growth and yield reductions were also considerably greater compared 
to the sum of individual effects of the pathogens (Khan and Akram, 2000). Similar 
results have been shown between M. incognita and F. oxysporum f sp. coffeae on coffee 
(Negron and Acosta (1989). On alfalfa, M. hapla increased the susceptibility of alfalfa to 
Phytophthora megasperma f. sp. medicaginis and increased the aggressiveness of the 
fungus and increased Phytophthora root rot and resultant plant mortality (Griffin et al., 
1993; Griffin and Gray, 1994). 

Antagonism: Gray et al. (1990) found that inoculated alfalfa seedlings were 
smaller after a single inoculation with only Phytophthora megasperma f sp medicaginis 
than after inoculation with both Meloidogyne hapla and P. megasperma f. sp. 
medicaginis. Their explanation for the disease suppression is that the nematodes feeding 
on immature roots may have interfered with infection by P. medicaginis. Valle-Lamboy 
and Ayala, (1980) reported an antagonistic interaction between root-knot nematodes M. 



19 

incognita and the fungus Pythium graminicola on sugarcane. Also, M. incognita was 
shown to protect Phaseolus vulgaris roots from the fungus Rhizoctonia solan i (Costa 
Manso and Huang, 1986). 

Papert and Kok (1999) reported that the gelatinous matrix in which eggs of root 
knot nematodes are deposited provide protection against microbial attack, possibly due to 
antibiotic compounds from the matrix or from associated bacteria. Orion and Kritzman 
(1991) studied the antimicrobial activity of gelatinous matrix of Meloidogyne javanica 
and concluded that it has antimicrobial activity probably for the protection of the 
nematode eggs. 

Additivity. It is commonly accepted that Meloidogyne arenaria increased the 
incidence of southern blight of peanut caused by the fungus Sclerotium rolfsii. However, 
Starr et al. (1996) showed in microplots that both pathogens are capable of reducing 
peanut yield, both reduce population growth of the other, and that no interaction occurs 
with respect to population growth, yield reduction, or incidence of southern blight. They 
concluded that the disease complex is due to additive effects of the pathogens on peanut. 
Whitney (1974) reported that the interaction between Pythium ultimum and Heterodera 
schachtii on root-rot of sugar-beet were synergistic but the interaction between P. 
aphanidermatum and H. schachtii was only additive. Abawi and Barker, (1984) showed 
that on tomato roots, necrosis increased with the population density of root-knot 
nematode M. incognita. They suggest that the presence of some soil-borne organisms 
such as Fusarium spp. that are found in association with the roots may increased damage 
caused by the nematodes, but the interaction was additive. An antagonistic interaction 
also occurs between the reniform nematode (Rotylenchulus reniformis) and the cotton 



20 

seedling blight fungus Rhizoctonia solani (Sankaralingam and McGawley, 1994). The 
presence of R. solani increased reproduction by R. reniformis, but the nematode did not 
increase cotton seedling blight. The combined effect of the nematode and the fungus 
inhibited cotton seedling blight compared to plant inoculated with only the fungus. 

Migratory Endoparasitic Nematodes: They are the nematodes that enter and 
migrate within the roots, feeding on various tissues. Feeding and migration of the 
nematodes damage root tissues resulting in necrotic extensive lesions on the root surface. 
The best documented example of a synergistic disease complex involving migratory 
endoparasitic nematodes and fungi is the association between Pratylenchus penetrans and 
Verticillium dahliae resulting in the potato early dying (PED) syndrome (MacGuidwin 
and Rouse, 1990; Powelson and Rowe, 1993). The disease causes premature vine death 
and declining yields of potato, and is a limiting factor in several potato production 
regions (Bird, 1981; Wheeler and Riedel, 1994). The effective control of this disease 
under field conditions with fumigants and nematicides has long suggested the 
involvement of plant parasitic nematodes. Results of experiments conducted in field 
microplots clearly showed a synergistic interaction between P. penetrans and V. dahilae 
resulting in PED syndrome. No measurable effect on symptom development and potato 
yield was obtained when P. penetrans was present alone, whereas light to moderate 
effects were observed with V. dahliae alone. Also, it was found that other species of 
Pratylenchus interact with Verticillium on PED in potato, but at a reduced level. A 
similar synergistic disease interaction occurs with F. oxysporium and Pratylenchus 
penetrans on alfalfa (Mouza and Webster, 1982). 



21 

Bowers, et al. (1996) reported that P. penetrans increased infection of potato roots 
by Verticillum dahliae. Potato roots were colonized by V. dahliae to a significantly 
greater extent when grown in soil infested with V. dahliae and P. penetrans than in soil 
infested with V. dahliae alone or with V. dahliae and P. crenatus. Infection by V. dahliae 
was not observed to be associated with the site of nematode feeding, and it is suggested 
that the effect of nematodes on initial infection may not be species-specific. It is 
concluded that the interaction between V. dahliae and P. penetrans in potato early dying 
may occur within the root early in the infection process, resulting in an altered or delayed 
host response to colonization by V. dahliae. 

Pratylenchus spp. are involved in root-roting complexes. Edmunds and Mai 
(1966) have shown that the combination between P. penetrans and Trichoderma viride 
cause more reduction in root and shoot growth in alfalfa and celery than in either 
organism alone. Santo and Holtezmann (1970) reported that simultaneous inoculation 
with P. zeae and P. graminicola reduced top and root growth more than either organism 
alone. Although both were present in the same lesion, effects of each appeared 
independent and additive. The most severe disease developed when plants were 
inoculated with nematodes seven days prior to inoculation with P. graminicola. Hasan, 
(1988) showed that root rotting symptoms in chrysanthemum roots caused by Pythium 
aphanidermatum and Rhizocotonia solani were increased in the presence of Pratylenchus 
coffeae and the interaction among the three organisms was synergistic. Similar results 
have been shown on maize, where the interactions among P. brachyurus, P. zeae and the 
root-rot fungus, Fusarium moniliforme were synergistic (Jordaan et al., 1987). LaMondia 
(1999) showed that P. penetrans alone or in combination with the black rot pathogen, 



22 

Rhizoctoniafragariae, reduced strawberry yield in microplots over time. The interaction 
of the two pathogens appeared to be additive rather than synergistic. 

Ectoparasitic nematodes: They are nematodes that remain outside the plant and 
penetrate with only a small portion of their body feeding either on surface tissues or on 
subsurface tissues. The sting nematodes, Belonolaimus spp, cause little damage to the 
cortex of plants when feeding, but have been associated with increased Fusarium wilt 
incidence in cotton. Meloidogyne incognita, Hoplolaimus galeatus and two population of 
B. longicaudatus (NC and GA) were introduced singly and in various combinations with 
Fusarium oxysporium. f. sp. vasinfectum on wilt susceptible Rowden cotton. Among the 
nematodes used, the combination of NC population of B. longicaudatus with Fusarium 
promoted the greatest wilt development. The combination of either population of B. 
longicaudatus with M. incognita and Fusarium induced greater wilt development than 
comparable inoculum densities of either nematode alone or when H. galeatus was 
substituted for either of these nematodes (Yang et al., 1976). Belonolaimus gracilis 
increased wilt incidence in the wilt susceptible cv. Rowden, and also in cv. Coker 100W, 
which exhibits a degree of wilt resistance (Holdeman and Graham, 1954). 

Mechanisms of Interactions between Nematodes and Fungi 

Interactions between nematodes and fungi are often indirect and occur due to 
induced modifications in the host plant. These interactions commonly result from either 
wounding or physiological alternations (localized or systemic) in the host plant. 
Nematode parasitism of plants requires wounding of the hosts, either by simple 
micropuncture or by rupturing or separating the plant cells (Taylor, 1979). Ectoparasitic 
nematodes may cause micropunctures on the plant root surface. Migratory endoparasitic 



23 

nematodes produce lesions in the root cortex and epidermis. Sedentary endoparasitic 
nematodes wound the host as second stage juveniles migrate intercellularly through the 
cortex and establish contact with vascular tissue to induce giant cells or syncytia. 

It has been suggested that the ectoparasitic Belonolaimus longicadatus has a very 
long stylet which may provide access to the vascular tissue of the plant to the wilt 
pathogen, in contrast to Hoplolaimus galeatus, which feeds in the cortical tissues. 
Fusarium wilt of cotton was enhanced by the ectoparasitic sting nematode B. 
longicadatus, but not by H. galeatus (Prot, 1993). Wounding may function to provide an 
undefended entry point for the fungus. However, complex anatomical, physiological 
changes in plant cells are associated with feeding by ectoparasitic nematodes. It is 
unknown how those changes affect associated parasites. Moreover, wounding likely 
increases leakage of root contents, which may be an attractants to other pathogens. 

Westerlund et al. (1974) observed that Fusarium oxysporium f sp. ciceri may 
require wounding for efficient infection of chickpeas. Studies have been done to 
compare the effects of inoculum levels and wounding on pathogenicity of F. oxysporium 
and F. solani. Application of 1 x 10 6 F. oxysporium conidia/ml onto seeds at sowing or 
onto young seedlings growing in U.C. mix caused disease in only three of 80 plants; 
these plants wilted and the fungus was recovered from their stems. If seedling roots were 
trimmed and dipped into conidial suspensions containing from 1 x 10 4 to 1 x 10 6 
conidia/ml, symptoms developed and the fungus was reisolated. When 1 x 10 4 to 1 x 10 6 
F. solani conidia/ml were applied to intact seeds or nonwounded seedlings, as described 
above, typical black rot lesions developed. Regardless of the physiological changes that 
may have been induced by these treatments, the study suggests that F. oxysporium 



24 

requires a wound for efficient infection whereas F. solani does not. 

On the other hand, Van Gundy et al. (1977) reported that the development of 
infection on tomato plants by R. solani was delayed by three to four weeks when the 
fungus was inoculated simultaneously rather than following inoculation with hi. 
incognita. The delay in predisposition of plants to fungal diseases by root-knot 
nematodes suggests that these nematodes are not just wounding agents facilitating the 
penetration of the fungi within the roots. Rather they showed that increased nutrient 
mobilization particularly nitrogenous compounds in root leachates three to four weeks 
after nematode infection, is favourable for maximum virulence of the fungi. When root 
leachates of plants inoculated simultaneously with the nematode and the fungus were 
permanently removed, no root-rot occurred. In contrast, when root leachates were not 
removed a severe root-rot developed. Moreover, when root leachates produced by M. 
incognita-infected plants were applied to roots of plants inoculated with R. solani alone, 
severe root-rot developed, whereas roots inoculated with R. solani receiving root 
leachates from control plants were free of decay. 

The mechanism of the interaction between Pratylenchus spp. and Verticillium 
spp. is related to the development of necrotic infection courts and biochemical changes in 
the attacked plants. Necrotic lesions on roots serve as infection courts which facilitate 
the establishment of the fungus in the court and subsequent invasion (Conroy et al., 
1972). Verticillium dahliae showed a distinct preference for the lesions on eggplant roots 
caused by P. penetrans. According to Conroy et al., (1972), lesions in tomato roots were 
more important for fungal invasion than any general physiological changes. The 
incubation period of Verticillium is shortened in Pratylenchus-inkcted plants (Bergeson, 



25 



1963; Faulkner et al., 1970). Pratylenchus minyus was shown to enhance susceptibility 
of peppermint to Verticillium wilt at the optimum temperature for the nematode (Faulkner 
and Bolander, 1969). Therefore, physical and physiological changes in the plants 
infected with Pratylenchus are collated together to enhance susceptibility of plants to 
Verticillium wilt. 

The mechanism of the interaction between sedentary endoparasitic nematodes and 
fungi is extensive and complex. For instance, the overall scenario of interactions 
between root-knot nematodes and Fusarium spp. can be visualized in different stages 
(Walter, 1965; Bergeson, 1972; Cook and Baker, 1983). The initial phase of the 
interaction occurs in the rhizosphere, where root exudates from root-knot infected plants 
stimulate the fungal pathogen. The exudates also, suppress the activity of actinomycetes 
which are antagonists of the wilt fungus. The next phase in these interactions involves 
the effect of root-knot infection on penetration of wilt fungi. Initially, it was thought that 
micropunctures caused by nematodes on the plant root facilitated entry of the fungal plant 
pathogens. Later, it was demonstrated that severity of fungal induced wilt diseases 
increased when root-knot nematodes were added three to four weeks prior to fungus 
inoculation of the host in comparison to simultaneous inoculations of both pathogens. 
This is led to the supposition that the nature of interactions between root-knot nematodes 
and Fusarium wilt fungi are physiological rather than physical. The ultimate phase of the 
interactions between the two pathogens occurs during the pathogenesis of the wilt fungus. 
Modifications of the host plant by root-knot nematodes are the key factor to this phase of 
interaction leading to increased wilt severity. 



26 



Root exudates reflect the biochemical and physiological changes induced by 
nematode infection, and hence the development of fungi in roots is influenced by the 
presence of additional metabolic products in the rhizosphere and on the roots. Root 
exudates, known to attract the motile stage of fungal pathogens (Zentmyer, 1961), 
represent a source of nutrients for soil microflora, and may be a stimulus for the 
germination of dormant fungal spores. Thus, change induced by nematodes in the root 
exudates may be the first stage in the synergistic interaction between nematode and fungi 
(Taylor, 1990). 

Several instances are known in which nematode galled tissue is more susceptible 
to fungi than non-galled tissue. Innovative work by Golden and Van Gundy, (1975) 
showed that galled okra and tomato roots infected with Meloidogyne incognita in the 
field and greenhouse were highly susceptible to infection by Rhizoctonia solani. Root 
decay by the fungus occurred 4-5 weeks after nematode infection. The sclerotia of R. 
solani formed only on galled tissue of okra and tomato roots infected with M. incognita. 
A modification of the cellophane-bag technique (Kerr, 1956) was used to study the 
prepenetration response of/?, solani to stimuli originating from hi. incognita-infected and 
control roots in situ. This technique allowed diffusable substances to pass through the 
membrane, but physically separated the fungus and host. Rhizoctonia solani responded 
to stimuli which originated from M. incognita-infected roots and passed through 
semipermeable cellophane membranes, by forming black sclerotia on the surface of the 
cellophane membranes directly opposite galls induced by M. incognita, while ungalled 
portions of nematode-infected control roots remained free of sclerotia. The results 
suggested that the mechanism by which nematode attack predisposed roots to secondary 






27 



invasion by R. solani was an indirect one. It is hypothesized that the leakage of nutrients 
from the roots was responsible for attracting the fungus to the galls, and for initiating the 
sclerotium formation. 

This hypothesis supported the work made in 1972 by Golden and Van Gundy 
which demonstrated that M. incognita infected tomato roots start leaking electrolytes 5 
days after invasion by the nematode, reaching a maximum level after 3 to 4 weeks. Such 
exudate-leakage contains more carbohydrates than proteins or amino acids, which 
increase in concentration only after two weeks or so. These exudates are known not only 
for enhancing growth of fungi like R. solani, F. oxysporium f. sp lycopersici, 
Thielaviopsis basicola, etc, but also have the ability to stimulate germination of the 
dormant fungal spores present in the soil environment. Also, fungal species like 
Trichoderma, Penicillum, Curvularia, Aspergillus, etc, which are generally considered 
weak pathogens become significant in the presence of nematode populations. 
Khan and Muller, (1982) reported that Rhizocotonia solani preferred M. hapla-induced 
galls on radish. The mycelium accumulated over them showed vigorous growth and 
abundant sclerotial formation. Extensive necrosis of the galls occurred and roots became 
obliterated. Non-galled regions of roots did not show sclerotial formation. 

Wang and Bergeson (1974) suggested that changes in total sugars and amino 
acids of M. incognita-infected root leachates contribute to predisposition of tomato plants 
to Fusarium wilt. During the first 14 days after nematode infection, when carbohydrates 
were abundant and the C/N ratio was high in M. incognita-infected root leachates, R. 
solani growth was stimulated in the rhizosphere and the fungus was attracted to the roots. 
Between 14 and 28 days following nematode infection, the C/N ratio decreased and this 



28 

low C/N ratio appears to favor parasitic development of R. solani (Weinhold et al., 1972). 

Although biochemical modifications of root leachates induced by root-knot 
nematodes appear to enhance the colonization of the rhizosphere by pathogenic fungi, 
and to attract and favor their growth to gall tissues, they also appear to lower the numbers 
of actinomycetes, antagonistic to F. oxysporium f. sp. lycopersici, in the rhizosphere. 
Bergeson et al. (1970) observed a highly significant reduction of actinomycetes and a 
significant increase in number of Fusarium propagules in the rhizosphere soil 
surrounding roots inoculated simultaneously with M. javanica and F. oxysporium f. sp. 
lycopersici compared to those observed when the fungus was inoculated alone. A similar 
observation was made by Noguera and Smits (1982) who suggested that the reduction in 
number of actinomycetes antagonistic to Fusarium in the rhizosphere of M. incognita- 
infected plants may be partly responsible for enhancement of pathogenic effect of the 
fungus. 

Modifications in hosts that enhance host susceptibly to fungal pathogens may 
involve systemic physiological changes. These changes render sites removed from the 
nematode infection more susceptible to the fungus. Adopting split-root techniques, 
Bowman and Bloom (1966) studied the breaking of Fusarium resistance in tomato 
cultivars Rutgers and Homestead by M. incognita. One part of the root system was 
inoculated with F. oxysporium f. sp. lycopersici and the other with M. incognita. They 
observed that wilt incidence was increased when the nematodes and the fungus were 
inoculated on opposite halves of the root system. Similar results were reported for the 
interaction between M. incognita and F. oxysporium f. sp. lycopersici by El-Sherif and 
Elwakil (1991) with the tomato cv. Tropic. Carter (1981) reported additive combined 



29 

effects of M. incognita inoculation on the roots and hypocotyl wounding, facilitating the 
penetration of R. solani on the severity of seedling disease of cotton. Because the two 
organisms infected spatially separate tissues, this additive effect indicated a localized 
effect from hypocotyl wounding but a systemic effect from the presence of M. incognita. 

Sidhu and Webster (1977) studied the translocation of the nematode's 
predisposing effect by bending over the stems of Fwsarw/M-resistant tomato plants (cv. 
Chico III) four times to produce four adventitious root systems in addition to the primary 
root system. They inoculated the M. incognita on the primary root systems and the 
fungus F. oxysporium f. sp. lycopersici on one of the root systems (primary or one of the 
four adventitious). When the fungus was inoculated on the primary root system, wilt 
symptoms were observed on the entire plant. When the fungus was inoculated on one of 
the adventitious root systems, wilt developed at the site of fungal inoculation and on the 
portion of the plant between the site of fungal inoculation and the apex of stem, whereas, 
wilt symptoms were minimal between the fungal inoculation site and the base of the 
plant. These results indicated that a predisposition factor produced or induced by the 
nematode can be transmitted at considerable distance from the nematode infection site to 
the upper foliage. Similar results were obtained by Hillocks (1 986) in an experiment in 
which plants growing in Meloi dogyne-infested soil were stem inoculated with the wilt 
fungus. The nematode increased wilt severity, despite the physical separation of the two 
organisms, indicating a systemic effect of the nematodes on the hosts resistance to fungi. 

Contradictory results were obtained in split root experiments when the nematode 
and the fungus were inoculated on opposite parts of the root system (Hillocks, 1986; 
Moorman et al., 1980). They did not observe any translocatable influence of M. 



30 

incognita on the development of F. oxysporium f. sp. vasinfectum and F. oxysporium f. 
sp. nicotianae on cotton and tobacco, respectively. However, they observed wilt increase 
and an enhancement of fungal infection when the nematode and the fungus were together 
on the same half-parts of the root system. These apparent contradictory results seem to 
indicate that the nematodes may have two effects favoring fungal infection of their hosts: 
i) a systemic effect where inhibition of host resistance mechanisms occurs, resulting in a 
stimulation of the fungal development in tissues not infected by nematodes, and ii) 
localized effects, where the fungus penetration and initial development in the host is 
enhanced by the modifications induced by the nematodes at their feeding sites. 

Disease Complexes Involving Citrus Nematodes and Fungi 

Several studies exist of the associations between nematode parasites of citrus and 
other microorganisms. Cobb, 1914, was the first to observe that other organisms may be 
associated with citrus nematode and cause a disease syndrome. The synergistic 
association between Tylenchulus semipenetrans and Fusarium solani has been shown by 
Thomas, 1923. F. solani can reduce growth in citrus seedlings under controlled 
conditions and the growth suppression of citrus seedlings caused by T. semipenetrans and 
F. solani together was greater than that caused by either organism alone (Van Gundy and 
Tsao, 1963). Root decay in lemon by Fusarium solani is increased significantly when T. 
semipenetrans is present at 30 °C but not at 20 °C or 25 °C; however, this does not hold 
true for F. oxysporium (O'Bannon et al., 1967). Labuschagne et al. (1989) showed that 
the combination of T. semipenetrans and F. solani only affected the growth of citrus trees 
under field conditions where the average maximum soil temperatures are below 30° C. 
The effect of both pathogens individually or in combination, would be significantly 



31 

greater where trees were subjected to stress caused by conditions such as waterlogging, 
low quality irrigation water, drought and other adverse conditions. 

Effects of Fungus on Nematode Populations 

In most synergistic interactions involving nematodes and fungi, usually fungi 
suppress the development and reduce the population density of the nematode. 
Interactions between the migratory endoparasitic nematodes such as Pratylenchus spp. 
and pathogenic fungi showed that the population of the nematode usually increases and in 
some cases decreases. Jordaan et al. (1987) found more root lesion nematodes 
Pratylenchus brachyurus in maize roots infected by Fusarium moniliforme which 
increased attraction and penetration of the nematodes into the roots. Hasan (1988) 
showed that the reproduction of lesion nematode Pratylenchus coffeae on 
chrysanthemum roots was decreased in the presence of Pythium aphanidermatum and 
increased in the presence of Rhizoctonia solani, however when both fungi were present 
nematode population was unaffected. Santo and Holtzmann (1970) reported that the 
normal populations of Pratylenchus zeae are much lower when Pythium graminicola is 
present than in fungus free roots. In contrast with migratory endoparasities, the sedentary 
endoparasites such as Meloidogyne spp., Heterodera spp. and Globodera spp. showed 
reduced population density in the presence of wilt fungi (Fusarium, Verticillum) and also 
with root-rot fungi (Pythium, Rhizocotonia and Phytophthora) (Powell, 1971; Hasan, 
1984; Gray et al., 1990). Jorgenson (1970) found that the interaction between 
Heterodera schachtii and F. oxysporium was antagonistic to the nematode. Damage to 
sugarbeets was less when the fungus and the nematode were present than when only the 
nematode was present. The fungus inhibited the nematode invasion and development on 



32 

sugarbeet seedlings. Three times more of//, schachtii larvae invaded sugarbeet seedlings 
that were inoculated with nematode only than invaded seedlings inoculated with both 
organisms. Anwar et al. (1996) showed that greater inhibition of root penetration by M. 
incognita, development of females, galling and nodulation occurred with simultaneous 
inoculation of R. solani and M. incognita. Nematode inoculation prior and after fungus 
treatment resulted in only slight to moderate inhibition of galling, nodulation and female 
development. Simultaneous inoculation of the fungus with nematodes exhibited a linear 
decrease in the final population of nematodes in soybean. Sakhuja and Sethi (1986) 
showed that both Fusarium solani and Rhizoctonia bataticola have an antagonistic effect 
on multiplication of M.javanica. Both fungi reduced nematode galling. Simultaneous 
inoculation of one or both fungi in combination with nematode was more inhibitory to 
galling in comparison to inoculations proceeding or succeeding the nematode. R. 
batiticola inhibited gall formation and nematode multiplication to a greater extent 
compared to F. solani. The effect could be possibly attributed to deleterious effects of 
metabolites of both fungi on juveniles. 

Nematode and Rhizosphere Microbial Community Structure 

Many biotic and abiotic factors affect the colonization of roots by microorganisms 
(Howie, 1985., Howie et al., 1987., Azad et al., 1985., Atkinson et al., 1975). One of the 
most limiting factors is the indigenous microorganisms, which may enhance or suppress 
root colonization by another organism (Weller, 1983., Brown, 1981). The presence and 
the infection by the nematode in the rhizosphere can significantly modify the rhizosphere 
environment and affect other rhizosphere microorganisms. The nutrient value of the 
gelatinous egg matrix and leakage from the roots due to the infection by the nematode 



33 

may be responsible for increasing or decreasing microbial density around nematode 
infected roots. 

Maloney et al. (1997) reported that successional changes in the composition of 
rhizosphere microflora occur in response to alteration in root physiology and microbial 
composition. Bowen and Rovira (1976) showed that nutrients, rather than space, are the 
limiting factor in competition among rhizosphere microorganisms during colonization, 
and that bacteria tend to congregate in grooves between cells where nutrients are most 
abundant. Indigenous microorganisms may also enhance colonization by introduced 
bacteria (Brown, 1981; Vojinovic, 1973; Weller, 1983). In another case, the population 
of indigenous, gram-negative bacteria of the Pseudomonas spp. were larger on roots 
infected by Gaeumannomyces graminis var tritici than on healthy plants (Weller, 1986). 
Rovira and Wildermuth, (1981) in electron microscopy studies showed that the bacteria 
proliferate in the lesions, probably owing to the greater availability of nutrients in these 
micosites. There are many of the rhizosphere bacteria involved in nematode disease 
complexes (i.e. Bacillus spp.; Burkholderia spp.; Arthrobacter spp.; Stenotrophomonas 
spp.; and others). Some of these bacteria are known as a bicontrol agents against plant 
pathogenic fungi and also nematodes or function as Plant Growth Promoting 
Rhizobacteria (PGPR). 

Bacillus megaterium forms spores that are resistant to unfavorable conditions, and 
is a good root colonizer, rhizosphere competitor and remains viable for extended periods, 
some of the multiple effects of B. megaterium on soil microorganisms have been studied 
(Liu and Sinclair, 1993). Bacillus megaterium is considered a potential biocontrol agent 
for Rhizoctonia root rot of soybeans (Zheng and Sinclair, 1996). Al-Rehiayani et al. 



34 

(1999) showed that the population densities of Meloidogyne chitwoodi and Pratylenchus 
neglectus were reduced up to 50% when potato plants were treated with B. megeaterium. 
Neipp and Becker (1999) showed that two strains of B. megaterium reduced nematode 
infection on sugarbeet when eggs were used as inoculum. Most of the strains they used 
in the experiment also reduced nematode egg hatch in vitro. 

Burkholderia cepacia and some other Bukholderia strains were given the name 
"Plant Growth Promoting Rhizobacteria" (PGPR) (Schroth and Hancock, 1981) because 
of their ability to colonize the roots aggressively and to improve plant growth and 
preempting the establishment of (or suppressing) (Deleterious Rhizosphere 
Microorganisms (DRMO) (Suslow, 1982). Burkholderia cepacia was used as a 
biocontrol agent for tomatoes against root diseases caused by R. solani and P. ultimum 
(Mao et al., 1998a) reduced Fusarium wilt (Larkin-Robert and Fravel-Deborah, 1998). 
B. cepacia reduced corn damping-off caused by species of Pythium and Fusarium (Mao 
et al., 1997 and 1998b). B. cepacia has been reported to colonize root hairs and enhance 
their development (De Freitas and Germida, 1990), to produce wide spectrum antifungal 
metabolites (Lambert et al., 1987), and to protect onion seedlings from damping off 
disease caused by F. oxysporium f. sp. cepae (Kawamoto and Lorbeer, 1976). 
Burkholderia cepacia is antagonistic to both R. solani and Pythium spp. and has been 
used for biological control of diseases caused by these pathogens (Cartwright and 
Benson, 1995; King and Parke, 1993; Millus and Rothrock, 1997). Meyer et al. (2001) 
showed that B. cepacia (Bc-2 and Bc-F) and Trichoderma virens (Gl-3) significantly 
suppressed numbers of root-knot nematode eggs and juveniles on roots of pepper plants. 
Burkholderia cepacia (Bc-F) increased shoot dry weights of nematode infected plants, 



35 

compared to controls. The combination of the biocontrol agents did not result in a 
beneficial synergistic interaction, however the agents individually suppressed the 
nematode populations. 

The bacterium Stenotrophomonas maltophilia inhibits brown patch disease, 
caused by R. solani (Giesler and Yuen, 1998) and protects sugar beet from Pythium- 
mediated damping-off (Dunne et al., 1998). Single inoculations with either W81 or Fl 13 
S. maltophilia strains effectively prevented colonization of sugar beet seeds by Pythium 
spp. in soil microcosms. In the field use of both strains as co-inoculated applications 
proved to be equivalent to the use of chemical fungicides, which improved the protection 
of sugar beet against Pythium-mediated damping-off. 

Strains of Arthrobacter are capable of causing destruction of Fusarium and 
Pythium (Mitchell and Hurwitz, 1964). Lytic Arthrobacter isolated from tomato 
rhizosphere reduced the infection by Pythium debaryanum from 88% (when the fungus 
was alone) to 25% when the fungus and the bacteria were together (Mitchell and 
Hurwitz, 1964). A single take-all lesion can enhance the population of P. fluorescens 
(Ahmad and Baker, 1987; Howie and Echandi, 1983) 10 fold per centimeter m of root. 
This is of practical significance, since the infected tissues are where the inhibitory 
bacteria are needed the most. Colonization of lesion provides considerable protection 
against secondary spread of take-all fungus on the roots. 

Mechanisms by which rhizobacteria exhibit biological control against soil 
pathogens have been reported (Kloepper, 1993) to include, antibiosis (through bacterial 
production of antifungal compounds including antibiotics and hydrogen cyanide 
(Brisbane et al., 1987; Thomashow and Weller, 1988; Howie and Suslow, 1991), 



36 

competition for nutrients or for ferric irons (Suslow, 1982; Weller, 1985; Elad and Chet, 
1987), competition for infection sites, (Osburn et al., 1989; Baker, 1968) or parasitism 
and production of lytic enzymes (Mitchell and Alexander, 1961; Mitchell and Hurwitz, 
1965; Sneh, 1981). 

Plant Growth Promoting Rhizobacteria prevented DRB from colonizing sugar 
beet because the PGPR occupy and exclude DRB from the cortical cell junctions, at 
which exudation of nutrients is maximal (Suslow, 1982). Elad and Chet (1987) reported 
that the suppression of pythial damping-off disease caused by Pythium aphanidermatum 
was correlated significantly to the competition for nutrients between germinating 
oospores of P. aphanidermatum and the biocontrol rhizobacteria. Osburn et al, (1989) 
showed that Psudomonas putida strain R20 that colonized the pericarps, seeds and roots 
of sugar beet had no effect on germination of Pythium ultimum sporangia in vitro. 
However in soil tests the bacteria delayed the fungal colonization of the pericarp 4 to 12 
hour after planting, by 24 hour, 90% of untreated seeds were infected with Pythium, 
whereas seeds treated with R20 showed 37% infection. The prevalence of damping off 
disease was 50% less than control after treatment with R20. They conclude that the 
disease suppression was due to the protection of the pericarps by occupation of pathogen 
infection sites by the bacteria. Arthrobacter and other rhizosphere bacteria that produce 
fungal cell wall lytic enzymes have shown a biological control activity against Pythium 
and Fusarium spp. However these bacteria did not colonize the roots suggesting that 
they may not be rhizobacteria but the biological control activity occurred due to 
parasitism of the fungi (Mitchell and Alexander, 1961; Mitchell and Hurwitz, 1965; 
Sneh, 1981). 



37 



Plant Growth Promoting Rhizobacteria can induce alteration in plant physiology 
resulting in increased host plant defense to pathogens attack (i.e induced resistance). Wei 
et al. (1991) showed that PGPR suppressed the anthracnose disease on cucumber caused 
by Colletotrichum orbicular by colonization of roots. The fungus was not detected in 
petioles or protected leaves suggesting that the antagonism or the competition for disease 
suppression was due to induced systemic resistance by applied PGPR as a seed 
treatments. Similar studies by Van Peer et al. (1991) who reported that Fusarium wilt of 
carnation was significantly reduced by root bacterization with Pseudomonas strain WC 
541 7r. The reduction occurs only when plants were bacterized 1 week before stem 
inoculation with F. oxysporium f. sp. dianthi and not when they received both treatments 
simultaneously. The bacterium was not detected in stem suggesting that the disease was 
suppressed not due to competition but due to induced resistance. 

Also, added bacteria may modify the rhizosphere environment and indirectly 
affect the plant growth. Bacillus megaterium and other Bacillus spp. known 
commercially as phosphobacterin (Copper, 1959; Mishustin, 1963) increased the vigor of 
wheat in the greenhouse but not in the field (Broadbent et al. 1977; Burr et al. 1978). It 
has been suggested that Bacillus spp. enhanced plant growth by different ways (i.e 
production of biologically active substrates (auxins and gibberellines) or transformation 
of unavailable minerals and organic compounds and make it available to the plant 
(Broadbent et al. 1977). 









CHAPTER 3 

INFECTION OF CITRUS ROOTS BY TYLENCHULUS SEMIPENETRANS REDUCES 
ROOT INFECTION BY PHYTOPHTHORA NICOTIANAE 

Introduction 

Citrus is one of the most economically important crops in many regions with 
Mediterranean and subtropical climates. The most commonly encountered association 
between nematodes and fungi that are pathogenic to citrus occurs between Tylenchulus 
semipenetrans Cobb and Phytophthora spp. The citrus nematode, T. semipenetrans, is 
distributed throughout all citrus growing regions of the world and causes the disease 
"slow decline" that results in significant reduction in fruit yield and size (Duncan and 
Conn, 1990). The nematode is a semi-endoparasite of the cortical cells of citrus fibrous 
roots; the female induces several nurse cells surrounding the head in the root cortex, 
while the posterior part of the nematode, including the egg mass, remains exposed in the 
soil (Van Gundy, 1958, Cohn 1965). Various species of Phytophthora also attack the 
citrus fibrous root cortex, causing a disease known as fibrous root rot (Graham and 
Menge, 1 999). The most commonly encountered species in the subtropics is P. 
nicotianae Dastur Breda de Haan (synonym = P. parasitica) (Graham and Timmer, 1992; 
Hall, 1993). Fibrous root rot is characterized by soft, water-soaked lesions that expand 
and quickly result in sloughing of the cortex to leave only the threadlike vascular 
cylinder. Significant loss of fibrous roots due to infection by P. nicotianae can result in 
less fruit yield and smaller fruit, similar to effects caused by T. semipenetrans (Graham 
and Menge, 1999). 

38 

. 



39 

A highly significant inverse relationship between numbers of nematodes following 
nematicide treatments and numbers of propagules of P. nicotianae in the soil was 
detected in field trials designed to evaluate the effects of nematicides and fungicides on 
concomitant populations of P. nicotianae and T. semipenetrans in a citrus grove in 
Florida (Graham and Duncan, 1997). Because fibrous root density was not increased by 
nematode management, the relationship between the population densities suggested the 
possibility that the nematode may inhibit population development of the fungus. We 
have subsequently investigated the nature of the interaction between P. nicotianae and T. 
semipenetrans in a series of field surveys and laboratory studies. In this chapter, we 
report results of experiments designed to determine whether infection of citrus fibrous 
roots by T. semipenetrans can modulate root infection by P. nicotianae and virulence of 
the fungus to citrus seedlings. 

Materials and Methods 
Phytophthora nicotianae infection of nematode-infected and noninfected root 
segments in vitro. Two bioassays were conducted to determine whether infection of 
roots by T. semipenetrans affects subsequent root infection by P. nicotianae. Nematode 
infected citrus roots were collected from naturally infected trees in the field and cut into 
(2-2.5 mm) segments that were either infected by a single female T. semipenetrans or not 
infected. Segments of each type were surface sterilized for 8 minutes in cupric sulfate 
(1,000 ppm) and then rinsed five times (500 cm 3 exchange of volume each time) in sterile 
distilled water. Phytophthora nicotianae isolate P-117 (obtained from citrus roots by J. 
H. Graham at Citrus Research and Education center in Lake Alfred, Florida) was cultured 
and maintained on Phytophthora-selective PARP-H medium, (a cornmeal agar amended 



40 



with antibiotics) (Graham, 1990; Mitchell and Kannwischer-Mitchell, 1992). Four agar 
plugs (5mm diameter) were removed with a cork borer from the margins of actively 
growing P. nicotianae colonies and placed on 2% water agar medium in 100 xl5 mm 
Petri dishes, equidistance apart. Six nematode-infected root segments were placed 2 mm 
from each agar plug (24 segments per dish). Each egg mass, exposed on the root surface, 
was placed in contact with the water agar facing the fungus plug. The same procedure 
was repeated with root segments not infected by the nematode. Each treatment was 
replicated six times. Root segments selected were of similar diameter and color. At 4, 7, 
and 1 1 days, root segments were removed from two dishes of each treatment (48 
segments per treatment) and placed on PARPH media for 72 hour to determine infection 
by P. nicotainae. The experiment was repeated, but segments were evaluated after 12 
days exposure to P. nicotianae. 

Effects of citrus nematode on fungal growth in roots and fungal virulence to 
citrus seedlings. Two whole-plant experiments were conducted to determine the effect 
of T. semipenetrans on the epidemiology of root infection by P. nicotianae. In both 
experiments, Sour orange (Citrus aurantium L.) seeds freshly removed from fruit were 
air-dried. After seed coats had been removed, seeds were surface sterilized with 10% 
commercial bleach (0.6% NaOCl) containing 0.01% Tween-20 for 10 minutes and then 
rinsed five times in sterile distilled water. A single sterilized seed was placed in a 2-cm 
deep depression made in the center of the surface of autoclaved soil mix (50:50 by 
volume, Candler fine sand (uncoated, hyperthermic Typic Quartzipsamments and 
shredded Canadian sphagnum peat moss (Scotts Inc., Sandusky, OH., U.S.A.) in 150 x 
25 mm glass test tubes. 



41 

In the first experiment, seedlings were inoculated with two levels of the nematode 
and two levels of the fungus. Eight treatments were established, fungus high, fungus low, 
nematode high, nematode low, fungus high nematode high, fungus low nematode high, 
fungus high nematode low, fungus low nematode low. 

The second experiment was established in a similar manner, but with different 
treatments. The pH of the soil mixture for half the plants was adjusted from 4.5 to 7.0 by 
addition of 3 mL per tube of 10% calcium carbonate, to favor nematode infection. Four 
factorial treatments were established for each pH level in this experiment: i) nematode 
infected seedlings, ii) fungus infected seedlings, iii) seedlings infected by both organisms, 
and iv) seedlings infected by neither organism. Both experiments were run with 10 
single plant replicates per treatment, in a completely randomized design arranged in racks 
in front of a window and maintained at room temperature (25 ± 2 °C), with daily diurnal 
cycles of light. 

Inoculum of T. semipenetrans was obtained from naturally-infected roots from the 
field. Eggs, juveniles, and males were scrubbed from root surfaces and collected on 
74/25 /im pore nested sieves. Nematodes were further separated from soil and plant 
debris by sucrose centrifugation (Jenkins, 1964), then surface sterilized with cupric 
sulfate (1,000 ppm) for 30 minutes and rinsed five times with sterile distilled water. In 
the first experiment, a 10 ml mixture of 8,000 or 80,000 eggs and second-stage juveniles 
of T. semipenetrans (low and high inoculum level, respectively) were pipetted into four 
holes around the stems of each plant in treatments receiving nematodes. In the second 
experiment, 80,000 nematodes per plant were similarly inoculated. Nematode infection 
was established for 6 months before P. nicotianae treatments were added. 



42 

Zoospores of P. nicotianae were obtained from colonies of isolate P-117 as 
described previously. Plugs were placed into sterile 60 xl5 mm Petri plates containing 10 
ml of sterile half-strength V-8 broth prepared by mixing 1 10 ml of clarified V-8 juice 
with 890 ml of water, kept at room temperature in the dark for 4 days for mycelial 
growth, after which the V-8 broth was decanted and 10 ml of sterile distilled water was 
added and decanted twice. Plugs were then incubated in 10 ml sterile distilled water 4 
days in light at room temperature to produce sporangia. Plates were refrigerated 30 
minutes and returned to room temperature to liberate zoospores. Zoospore suspensions 
were decanted after 45 minutes combined and quantified using a hemacytometer 
(American Optical Co., New York, NY., U.S.A.). Low and high levels (9,000 and 
90,000 zoospores) of fungal inocula in 10 ml water were introduced via canula, 1 to 10 
cm deep into the soil in the tubes in the first experiment. Only the high level of 
zoospores (90,000) was used in the second experiment. Ten milliliters of sterile distilled 
water was added in the same manner to control tubes. 

Six weeks after fungal inoculation, soil was gently rinsed from tubes to remove the 
plants. Roots were blotted and tap and fibrous roots were separated and weighed. Stem 
and leaf fresh and dry weights were measured. Root systems from five plants per 
treatment were blender extracted (Duncan and El-Morshedy, 1996) to estimate the 
numbers of eggs, second-stage juveniles, and females per gram of root. Roots from the 
remaining replicates were gently rinsed with tap water and dried for 48 hour in the oven 
(70 °C), then ground by mortar and pestle. Concentration of P. nicotianae protein in 30 
mg dried roots was determined using the Agri-screen immunoassay kit (Enyzme Linked 






43 



Immunosorbent Assay (ELISA) for detection of Phytophthora (Neogen Corp., Lansing, 
ML, U.S.A). 

In the first experiment, The data from six balanced factorial treatments (fungus 
high, fungus low, fungus high nematode high, fungus low nematode high, fungus high 
nematode low, fungus low nematode low) (all containing the fungus) were analyzed with 
2-way ANOVA (Minitab Inc., State College, PA, U.S.A) in which fungus (high or low) 
and nematode (absent, high or low) were main factors. Data in the second experiment 
were analyzed by 3-way ANOVA in which pH, fungus and nematode were the main 
factors. 

Results 

Phytophthora nicotianae infection of nematode infected and non-infected root 
segments in vitro. In the first test, approximately twice as many root segments were 
infected by P. nicotianae in the absence of nematodes by 10 days post-exposure (Fig. 3- 
1) (P < 0.01). Results of the second test at 12 day post-exposure were consistent with 
those of the first. 

Effects of citrus nematode on fungal growth in roots and virulence to citrus 
seedlings: In both whole-plant experiments, infection by the citrus nematode reduced the 
fungal protein (as measured by ELISA) in the seedling roots and increased plant weight 
compared to plants treated only with the fungus. In the first experiment (Fig. 3-2), levels 
of fungal protein in the roots infected by both organisms were 53 to 65% lower (P < 0.05) 
than in roots infected by only the fungus. Compared to plants infected only by P. 
nicotianae, shoot weights were 33 to 50% greater (P < 0.05) in plants infected by both 
parasites. Fibrous and tap root weights were 5 to 23% and 19 to 34% greater (P < 0.05), 



44 

respectively, in nematode fungal combination treatments compared to the fungus alone. 
There was no significant effect of P. nicotianae on T. semipenetrans reproduction at any 
inoculum level. Mean (± standard error) nematode females per gram root was 130 ± 26 
in high inoculum treatments and 76 ± 10 in low inoculum treatments. Corresponding 
means for offspring per gram root were 1,953 ± 158 and 1,178 ± 198 (Table 3-1). 

In the second experiment, soil pH affected the growth of seedlings and the 
population growth of the nematode and the fungus. Root and stem fresh weights of 
untreated controls were 22% and 40% greater, respectively, when grown at pH 7.0 
compared to pH 4.5. The number of nematode females and offspring per gram of roots 
from plants inoculated only with nematodes were 3.6 and 13.2 times greater, respectively, 
at high pH compared to low pH (Table 3-1) (P < 0.05). 

Absorbance readings from ELISA plates to detect protein of Phytophthora sp. were 
72% greater at pH 7.0 than at pH 4.5 for plants inoculated only with P. nicotianae (Fig. 
3-3) (P < 0.05). Phytophthora nicotianae was the only parasite that reduced seedling 
mass at either pH. Compared to controls, P. nicotianae reduced root fresh weight by 
37% at pH 7.0 and 16% at pH 4.5 (P < 0.05). The fungus reduced stem fresh weight by 
27% at pH 7.0 and by 20% at pH 4.5 (P < 0.05). 

Nematodes and fungi each inhibited population growth of the other in some 
conditions. Tylenchulus semipentrans completely suppressed detection of P. nicotianae 
protein in root tissue at both pH levels (Fig. 3-3). At pH 7.0 which favored nematode 
infection, the fungus had no effect on the final population density of the nematode (Table 
3-1). However, in plants grown at pH 4.5, which was less favorable for nematode 
infection, the fungus reduced the number of nematode females and offspring per gram of 



45 

root by 39% and 58%, respectively (P < 0.05). 

With respect to seedling variables, the presence of the nematode completely 
inhibited disease caused by P. nicotianae (Fig. 3-3). Regardless of soil pH, there were no 
significant differences in stem or root fresh weights in plants infected by both organisms 
compared to untreated controls (Fig. 3-3). The infection by T. semipenetrans increased 
stem fresh weights (Table 3-2; Fig. 3-3). There was an interaction between the nematode 
and fungus in terms of root fresh weight (P < 0.005) and fungus protein in roots (P < 
0.001) (Table 3-2). Further one-way ANOVA revealed that the fungus reduced stem (P < 
0.02) and root (P < 0.002) weights in the absence of the nematode, but had no effect on 
stem (P < 0.97) or root (P < 0.25) weights in the presence of the nematode. 
Phytophthora nicotianae treatments resulted in increased (P < 0.001) fungal protein in 
roots, whereas fungal protein in the T. semipenetrans plus P. nicotianae treatment did not 
differ from that in the untreated control (Table 3-2; Fig. 3-3) (P < 0.618). 

Discussion 

An antagonistic interaction between T. semipenetrans and P. nicotianae in these 
experiments resulted in less infection of roots by the fungus, reduced fungal development 
in roots, and less growth reduction of citrus seedlings. Although nematode densities 
among treatments were manipulated by different inoculation rates and different 
conditions of soil pH, no density dependence in the antagonistic effects was observed for 
the range of nematode densities achieved in these experiments. For example, 
manipulation of pH resulted in an approximate 5-fold difference in infection rates by the 
nematode; however, infection by the fungus and subsequent disease were prevented by 






46 



the nematode under both pH conditions. The effect of pH is consistent with results of 
Van Gundy and Martin (1961) who found higher population densities of 
T. semipenetrans in alkaline than in acid soils. The pH optimum of soil for 
T. semipenetrans development is 6.0-7.5 (Van Gundy et al., 1964). 

The consistency of the antagonistic effect in these studies, and observations of 
significant increases in P. nicotianae propagule densities in soil following management 
of T. semipenetrans in a citrus orchard (Graham and Duncan, 1997), suggest that the 
interaction between this nematode and the fungus is potentially of economic significance. 

Our results differ from those of most research involving nematode-fungus 
interactions. When interactions occur, the nematode frequently plays the primary role as 
modifier of the host making it more susceptible or more suitable for other pathogens 
(Pitcher, 1978; Powell, 1979). Interrelationships between plant-parasitic nematodes and 
soil-inhabiting micoorganisms were first observed by Atkinson (1892) who noted a very 
close relationship between Meloidogyne spp. and Fusarium wilt fungi in cotton. The 
combinations of these pathogens always contribute to more severe losses from wilt than 
did the fungus alone. Various systems involving sedentary endoparasitic, migratory 
endoparasitic and ectoparasitic nematode- fungal association are well reviewed 
(Bergeson, 1972; Holdeman and Graham, 1954; MacGuidwin and Rouse, 1990; Mai and 
Abawi, 1987; Powell, 1971a, 1971b; Powelson and Rowe, 1993; Prot, 1993). Root-knot 
and cyst nematodes often predispose plants to more severe infection by other pathogens 
such as Fusarium spp., Phytophothora spp., and Rhizoctonia spp. (Carter, 1981; McLean 
and Lawrence, 1993; Powell et al., 1971; Roy et al., 1989; Webster, 1985; Whitney, 
1974). Sting nematode, Belonolaimus spp., which are ectoparasitic in their feeding habit 



47 

cause little damage to the root cortex when feeding, but yet they have been associated 
with increased Fusarium wilt incidence in cotton (Yang et al., 1976). Synergistic disease 
complexes involving migratory endoparasitic nematodes include the associations between 
Pratylenchus penetrans and Verticillium dahliae resulting in the potato early dying 
syndrome (Bowers, et al., 1996) and F. oxysporum and Pratylenchus penetrans on alfalfa 
(Mouza and Webster, 1982). 

Antagonism toward plant pathogenic fungi by nematodes is not unknown. Valle- 
Lamboy and Ayala, (1980) reported an antagonistic interaction between root-knot 
nematodes M. incognita and the fungus P. graminicola on sugarcane. The presence of 
the nematode in combination with the fungus interfered with the fungus development and 
the plants grew and developed better than when the two microorganisms act separately. 
Also, M. incognita has been shown to protect Phaseolus vulgaris roots from the fungus 
R. solani (Costa Manso and Huang, 1986). Gray et al. (1990) found that survival of 
alfalfa seedlings was reduced following inoculation with Phytophthora megasperma f. sp. 
medicaginis compared to inoculation with both M. hapla and P. megasperma. They 
suggested that the nematodes feeding on immature roots may have interfered with 
infection by P. medicaginis. An antagonistic interaction also occurs between the 
reniform nematode (Rotylenchulus reniformis) and the cotton seedling blight fungus 
Rhizoctonia solani (Sankaralingam and McGawley, 1994). The presence of/?, solani 
increased reproduction by R. reniformis, and the combined effect of the nematode and the 
fungus inhibited cotton seedling blight compared to plant inoculated with only the 
fungus. The similarity between results of the present study and that of Sankaralingam 
and McGawley (1994) is noteworthy because both T. semipenetrans and R. reniformis are 



48 

sedentary semi-endoparasites that infect roots by penetrating the mature epidermis and 
root cortex. This infection process is fundamentally different than that of numerous 
sedentary endoparasites that penetrate in the rapidly developing zone of elongation and 
for which antagonistic interactions with fungi are generally unknown. Thus, adaptation 
of mechanisms to prevent fungal infection of nematode feeding sites may be affected by 
the mode of root infection by nematodes. 

Both T. semipenetrans and P. nicotianae frequently reside concomitantly in the 
citrus rhizosphere; therefore, it is not surprising that the nematode may protect its feeding 
site and may interfere with the fungus either indirectly (through resource competition, 
alteration of host physiology or alteration of the microbial community in the 
rhizosphere), or directly (anti-fungal chemicals). Tylenchulus semipenetrans was shown 
to increase the incidence of Bacillus megaterium and Burkholderia cepacia in the citrus 
rhizosphere (El-Borai et al., 2000) and both bacteria inhibit a variety of soilborne plant 
pathogens (Al-Rehiayani et al., 1999; Mao et al., 1997; 1998a, 1998b; Millus and 
Rothrock, 1997; Zheng and Sinclair, 1996). Eggs of T. semipenetrans were recently 
found to inhibit mycelial growth of P. nicotianae and F. solani in vitro, in contrast to 
those of M. arenaria (El-Borai Kora et al., 2001). Thus, the nature of the interaction 
between T. semipenetrans and P. nicotianae may be complex and additional work on the 
subject is warranted. 

From a practical standpoint, the results of this study suggest that growers need not 
be concerned that citrus nematode will exacerbate yield or tree losses due to 
Phytophthora-induced root or crown rot. Indeed, infection by the nematode may mitigate 
damage by the fungus, although field studies are needed to determine whether this 



49 

interaction is economically significant and whether this interaction should form a basis 
for management tactics. 





















50 



"a . 

o a 

a « 

c.8 

.2 5 
o ^ 

8 * 



0.16 - 



0.12 



0.08 - 



0.04 



0.00 



o 


1 st 


test- 


no female 


# 


1 st 


test- 


female 


n 


pnd 


test 


- no female 


■ 


ond 


test 


- female 



6 



-i 1— 

4 5 



. 



6 



[] 



6 



—r- 

7 



8 9 10 11 12 

Days after exposure to P. nicotianae 



Figure 3-1. Phytophthora nicotianae infection of Tylenchulus semipenetrans 
infected and non-infected citrus root segments in vitro. 



51 



3.0 i 



o 



u 



o 

o c 

a. x> 
II 

PL, 




1 



•O fe) 



S 



1 



0.20 



0.15 - 



0.10 



0.05 - 



0.00 



0.5 



0.4 - 



b 



c/3 f3 



on 



I 0.3 





0.2 



0.1 



0.0 J 



FH FL FH FL FH FL 

NH NH NL NL 

Figure 3-2. Effect of Tylenchulus semipenetrans on growth and pathogenicity 
of Phytophthora nicotianae (FH=Fungus High, FL=Fungus Low, 
NH=Nematode High and NL=Nematode Low). Bars indicate the 
standard error of the mean for ten seedlings replications per treatment. 



52 



Table 3-1. Effect of different soil pH (low - 4.5 and high = 7.0) on rate of reproduction 
of the citrus nematode Tylenchulus semipenetrans. 



Females/gof root 



Offspring/g of root 



Treatment 



pH4.5 



pH7.0 



pH4.5 



pH7.0 



Untreated control 
Tylenchulus semipenetrans 
Tylenchulus semipenetrans 
+ Phytophthora nicotianae 



121.3 ±38.2 554.6 ±110.9 182.5 ±24 2583.9 ±562.9 
76.40 ±2.6* 563.9 ±152.3 77.7 ± 8.3* 2722.0 ± 688.2 



Data values are means of 6 replications ± standard error of the mean. 
* Indicates significant difference from control at P < 0.05. 



Table 3-2. Analyses of variance of effects of Tylenchulus semipenetrans on stem fresh 
weight, root fresh weight and Phytophthora nicotianae protein in citrus roots 
in thelaboratory 







Stem 


weight 


Root 


weight 


Fungal 


protein 




F-value 


P-value 


F-value 


P-value 


F-value 


P-value 










Tylenchulus semipenetrans 






PH 




37.80 


0.000 


4.39 


0.041 


0.64 


0.432 


Nematode 




5.09 


0.028 


4.93 


0.031 


22.76 


0.001 


Fungus 




2.55 


0.116 


1.12 


0.295 


22.71 


0.001 


pH x Nematode 




0.39 


0.534 


0.02 


0.889 


2.05 


0.165 


pH x Fungus 




0.09 


0.769 


0.25 


0.619 


2.31 


0.141 


Nematode x Funj 


JUS 


2.46 


0.122 


8.64 


0.005 


21.75 


0.001 


pH x Nematodex 


Fungus 


0.64 


0.427 


1.77 


0.190 


1.91 


0.179 



53 



Low pH=4.5 






High pH=7.0 



1.0 



<D 



U 



* 9 

S 

1 | 0.5 



0.0 
0.3 

0.2 

0.1 

0.0 
0.4 

0.3 



'53 



a 



o 3 

o 



op 
'53 

II 



£3 0.2 



E 

■4—* 

C/3 



0.1 



0.0 



Control 
Nematode 
Fungus 
Nematode 
+fungus 











CNF N+F 



1.0 



0.5 



0.0 
0.3 



0.2 



0.1 



0.0 
0.4 



0.3 



0.2 



0.1 



0.0 



o 



D 



o 5 

— TZ 

CX.O 
ca O 



3 
fe 



c3 



2?' 
'5 



a 



o w 



'53 



<d 



oo 



& 



C N F N+F 



Figure 3-3. Effect of pH and the citrus nematode Tylenchulus semipenetrans on growth in 
roots of Phytophthora nicotianae and damage by the fungus to citrus seedlings 
(C=Control, N=Nematode, F=Fungus, N+F=Nematode plus Fungus). Bars 

indicate the standard error of the mean for 10 seedlings replications per 

treatment. 



CHAPTER 4 

TYLENCHULUS SEMIPENETRANS ALTERS THE MICROBIAL COMMUNITY IN 

THE CITRUS RHIZOSPHERE 

Introduction 

The interactions between plant-parasitic nematodes and other plant pathogens are 
commonly perceived to be indirect, the result of modifications in the host plant such as 
localized or systemic responses to wounding (Carter, 1981; Hillocks, 1986; Moorman et 
al., 1980; Westerlund et al., 1974) or other physiological alterations (Bowman and 
Bloom, 1966; Van Gundy et al., 1977). Root exudates reflecting the biochemical and 
physiological changes induced by nematode infection alter microbial community 
dynamics in the rhizosphere and on the roots (Wang and Bergeson, 1974; Weinhold et 
al., 1972; Van Gundy et al., 1977). Root exudates attract the motile stage of fungal 
pathogens (Zentmyer, 1961), represent a source of nutrients for soil microflora, stimulate 
the germination of dormant spores, and may be the first stage in synergistic interactions 
between nematodes and fungi (Taylor, 1990). For example, root leachates induced by 
root-knot nematodes {Meloidogyne spp.) can enhance the colonization of the rhizosphere 
by pathogenic fungi (Golden and Van Gundy, 1972, 1975; Kerr, 1956; Khan and Muller, 
1982) and reduce the numbers of actinomycetes, antagonistic to other fungi in the 
rhizosphere (Bergeson et al., 1970). Chemicals that emanate directly from nematodes 
have been also shown to affect associated microorganisms. The gelatinous matrices 
surrounding eggs of the root-knot nematodes, M.javanica and M.fallax provide 



54 



55 

protection from microbial attack due either to antibiotic compounds of nematode origin 
or from associated bacteria (Orion and Kritzman, 1991; Papert and Kok., 1999). 

The most common association between pathogens of the citrus fibrous root system 
is likely that between the citrus nematode T. semipenetrans Cobb and the root rotting 
fungus P. nicotianae Breda de Haan (synonym = P. parasitica Dastur (Hall, 1993). Both 
organisms are nearly ubiquitous in citrus growing regions. Each feeds on the cortex of 
fibrous roots and both have been shown to reduce the density of the fibrous root system 
(Duncan et al., 1993; Graham and Menge, 1999). Previous work done in the field and 
greenhouse (Graham and Duncan, 1997) followed by a series of in vitro, laboratory, and 
greenhouse studies (El-Borai et al., 2000) showed that the citrus nematode T. 
semipenetrans reduced root infection by P. nicotianae and increased growth of citrus 
seedlings compared with seedlings infected by P. nicotianae alone. These results imply 
an antagonistic interaction between T. semipenetrans and P. nicotianae. 

Potential mechanisms for antagonism by nematodes to fungi include direct 
antibiosis, competition for resources in the roots, or indirect mediation through increased 
colonization of nematode feeding sites by microorganisms antagonistic to P. nicotianae. 
Following the latter hypothesis, we sought to determine whether infection by T. 
semipenetrans changes the composition of rhizosphere inhabiting microorganisms, and to 
identify microorganisms that are consistently associated with the nematode. We also 
conducted in vitro and whole plant experiments with candidate bacteria species to 
determine their capacity to inhibit root infection by P. nicotianae in the presence or 
absence of citrus nematode. 



56 

Materials and Methods 

Field survey. A survey was conducted in three citrus orchards to investigate 
whether root infection by T. semipenetrans is associated with changes in communities of 
bacteria and fungi in the rhizoshere. Roots naturally infected by T. semipenetrans were 
collected from citrus orchards near Ona, Bartow, and Lake Alfred in central Florida. The 
groves were separated by distances ranging from 1 8 to 45 miles. Five random trees were 
sampled in each orchard and five fibrous root samples (0-30 cm-depth) collected from 
each tree were composited. Six groups of 30 root segments (2.0-2.5 mm length) prepared 
from each sample were placed in 5-ml distilled water and macerated for approximately 8 
seconds in a Tissuemizer (Tekmar Co., Cincinnati, OH., U.S. A). Half of the groups 
contained root segments infected by female T. semipenetrans and the remaining groups 
contained only uninfected segments as identified at lOx magnification under a 
stereomicroscope. The resulting suspensions were diluted (10, 10" 2 , and 10" 3 ) and 0.1 -ml 
of each dilution was streaked on five Petri dishes each of two different media: PARP-H, 
corn meal agar amended with antibiotics, selective for Phytophthora (Graham, 1 990; 
Mitchell and Kannwischer-Mitchell, 1992), and potato dextrose agar (PDA; Difco 
Laboratories, Detroit, MI, U.S.A). After 72 hours, bacterial and fungal colonies were 
counted and individual colonies isolated. 

Bacteria were cultured on 1.5% nutrient agar (Sigma Chemical Company, St. 
Louis, MO, U.S.A). The isolates were identified by fatty acid analysis using fatty acid 
methyl-esters (FAMEs) (Sasser, 1990) and the Aerobic Bacterial Library of MIDI 
(Microbial Identification, Newark, DE) (Anonymous, 1996). Cultures were maintained 
at -80° C. Predominant fungal colonies were transferred to new nutrient agar plates 



57 

within glass cylinder cells for separation from contaminant organisms by allowing the 
fungus to grow downward and laterally. The plates were incubated at room temperature 
( 25 °C) for 5 days, after which the procedure was repeated. Fungi were identified to 
genus or species level by microscopic and sterioscopic analysis, using taxonomic keys. 

The survey was conducted twice, first in July 1998 at two sites (Lake Alfred and 
Ona), when the bacterial and fungal colony forming units (CFUs) were quantified and 
again in March 1999 at three sites, when CFUs were quantified and identified to species. 
Data for total bacteria CFUs in both surveys at Lake Alfred and Ona were analyzed by 
three-way analysis of variance in which site, root type and year were main factors. Data 
for each microorganism identified in the second survey (3 sites) were analyzed by two- 
way analysis of variance in which site and root type were main factors. 

In vitro inhibition of P. nicotianae by isolated bacteria. Bioassays were 
conducted to determine whether bacteria associated with T. semipenetrans-infccted roots 
affect growth of P. nicotianae. Bacterial isolates were prepared by streaking them onto 
nutrient agar and allowing the isolates to grow for 48 hours at room temperature. Using a 
bacterial loop, a single cell colony from newly grown colonies was transferred to nutrient 
agar in 100 x 15 mm Petri dishes (Fisher Scientific, Pittsburgh, PA, U.S.A) by streaking 
the cells in a circle (3-cm wide) circumscribing the center of the dish. A 4-mra mycelial 
plug taken from an actively growing colony of P. nicotianae (isolate P-l 17 obtained from 
citrus roots by J. H. Graham at Citrus Research and Education Center, Lake Alfred, 
Florida) grown in PARP-H media was placed in the center of the dish. Ten replicate- 
plates for each bacterium were incubated at room temperature for 3 days after which the 
P. nicotianae colony radius was measured from eight different directions and means 



58 

determined. Data were subjected to analysis using Dunnett's test (Dunnett, 1955) to 
compare the control treatment (P. nicotianae alone) with the five tested bacteria. 

Effects of nematode-associated bacteria on the interaction between 
T. semipenetrans and P. nicotianae. Two experiments (greenhouse and laboratory) 
were conducted to determine whether rhizosphere bacteria in the presence of absence of 
T. semipenetrans affect the virulence of P. nicotianae on citrus. Isolates of Burkholderia 
cepacia and Bacillus megaterium were used to establish the following incomplete 
factorial treatments: bacteria alone, bacteria + fungus, bacteria + fungus + nematode, 
nematode alone, fungus alone, nematode + fungus, and untreated control. All treatments 
were established under two different conditions of soil pH (low, 4.5 and high, 7.0). Both 
greenhouse and laboratory experiments were run with ten single plant replicates per 
treatment, in a completely randomized design. 

In the greenhouse experiment, sour orange {Citrus aurantium L.) seeds freshly 
removed from fruit were air-dried. Seed coats were removed and seeds were surface- 
sterilized for 10 minutes with 10% commercial bleach (0.6% NaOCl) containing 0.01% 
Tween-20, then rinsed five times in sterile distilled water. Sterile seeds were placed 
individually in 150 x 25 mm autoclaved capped tubes containing MT medium 
(Murashige and Tucker, 1969) solidified with 0.9% agar (Difco, Detroit, MI, U.S.A) and 
containing 3% sucrose (pH 6.2) where they germinated aseptically. The culture was 
maintained at (26 °C ± 2) with 16 hours of cool-white fluorescent light. 

Bacterial inocula were prepared (after three weeks storage at -80 °C) by streaking 
isolated bacteria onto nutrient agar 1.5% in 100 x 15 mm Petri plates and evaluating them 
for purity after incubation at room temperature for 48 hours. Single cell colonies from 



59 

each bacterial culture were transferred to sterile vials with 50 mL Nutrient Broth (Sigma 
Chemical Co., St. Louis, MO, U.S.A) and incubated with shaking for 36 hours. Cultures 
were transferred to sterile 50-ml tubes under aseptic conditions and centrifuged at (1000 
rpm for 10 minutes) to obtain a bacterial pellet. Pellets were resuspended in 15 mL 
sterile phosphate buffer (5.8 g Na 2 HP0 4 and 3.5 g KH 2 P(V1000 mL sterile distilled 
water, pH 7.2). The final suspension absorbance was measured for each bacterium in a 
spectrophotometer at 620 nm, and adjusted to an average population of 2.4 x 10 7 per mL. 
Bacterial suspensions were immediately pipetted into the tubes, 2ml per plant, and 2ml 
sterile distilled water was added to tubes not receiving bacteria. Three weeks after 
adding the bacteria, seedlings were transferred to plastic Ray- Leach Containers (1.5- 
diam. x 8.5 depth) (Stuewe & Sons Inc. Corvallis, OR.,U.S.A) with autoclaved soil mix 
(50:50 by volume, Candler fine sand (uncoated, hyperthermic Typic 
Quartzipsammentsand), and shredded Canadian sphagnum peat moss; Scotts Inc., 
Sandusky, OH, U.S.A). Plants were maintained in the greenhouse (22-24 °C with forteen 
hours light per day). Ten replications were used for each treatment. 

Inoculum of T. semipenetranswas obtained from naturally infected field roots. 
Eggs, juveniles and males were scrubbed from root surfaces by hand rubbing the roots 
together in water. The nematode life stages were collected on 74/25 pm pore nested 
sieves. Nematodes were then separated from soil and plant debris by sucrose 
centrifugation (Jenkins, 1964), surface sterilized with cupric sulfate (1000 ppm) for 30 
minutes, and rinsed with five exchanges (500 cm 3 volume each) of sterile distilled water. 
Two weeks after transplanting the seedlings, a mixture of 90,000 eggs and second-stage 
juveniles of T. semipenetrans were pipetted into four holes around the stems of each plant 






60 

in treatments receiving nematodes. Nematodes were permitted to establish on the 
seedling roots for 6 months before P. nicotianae treatments were added. 

Zoospores of P. nicotianae were obtained by removing nutrient agar plugs from 
actively growing colonies of P. nicotianae (P-l 17). Plugs were placed into sterile 60 x 
15 mm Petri plates containing 10ml sterile half-strength V-8 broth prepared by mixing 
1 10 ml of clarified V-8 juice with 890 ml of water. Plates were incubated in the dark at 
room temperature 4 days for mycelial growth, after which the V-8 was decanted and 
10ml of sterile distilled water was added and decanted twice. Plugs were then incubated 
in 10-ml sterile distilled water for 4 days in the light at room temperature to produce 
sporangia. Plates were refrigerated for 30 minutes and returned to room temperature to 
liberate zoospores. The zoospore suspensions were decanted after 45 minutes, combined 
and quantified using a hemacytometer (American Optical Co., New York, NY, U.S. A), 
and 90,000 zoospores in lOmL water were introduced via canula 1 to 10cm deep in soil 
of appropriate tubes. Ten-milliliter water was added in the same manner to tubes not 
receiving zoospores. 

The laboratory experiment was similarly established with the same treatments as in 
the greenhouse, except the experiment was conducted in 1 00-ml glass test tubes into 
which a single sterile decorticated seed was introduced, as described previously, and 
placed into the same autoclaved soil mix and allowed to germinate. The soil pH for half 
the plants was adjusted from 4.5 to 7.0 by addition of 3ml/tube of 10% calcium carbonate 
to favor nematode infection. 

Six weeks after fungal inoculation, soil was gently rinsed from the plastic Ray- 
Leach Containers or from the glass tubes to remove the plants. Roots were gently blotted 



61 

and tap roots and fibrous roots were separated and weighed. Stem fresh and dry weights 
were measured. Root systems from five plants per treatment were processed (Duncan 
and El-Morshedy, 1996) to estimate the number of eggs, second-stage juveniles, and 
females per gram of root. Serial dilutions of macerated root suspensions were made from 
each treatment. A 300-ul aliquot from each dilution was plated onto nutrient agar to 
determine numbers of colony forming units (CFUs) of bacteria. Bacteria isolates were 
identified as described previously. Roots from the remaining replicates were washed free 
from adhering soil with a minimum of tap water, dried for 48 hours in the oven (70 °C) 
and ground with a mortar and pestle. Concentration of P. nicotianae fungal protein in 30 
mg/samples was determined by ELISA test using the Agri-screen Phytophthora detection 
immunoassay kit (Neogen Corp., Lansing, MI, U.S. A). Subsets of the data from both 
experiments were analyzed by two-way ANOVA (Minitab Inc., State College, PA, 
U.S.A) of balanced factorial treatments (all those which contained P. nicotianae). Main 
factors were pH and treatments. Population data were transformed (loge n+1) prior to 
subjected to ANOVA, but un trans formed means are reported. 

Results 
Field survey. The number of bacteria CFUs from T. semipenetrans-inkcted 
roots was greater than from uninfected roots in all groves surveyed (Tables 4-1,4-2). 
Analysis of variance of repeated surveys for two locations (Lake Alfred and Ona) showed 
highly significant differences between surveys, groves, and type of root segment (Table 
4-1). In the second survey, conducted at three sites, Bacillus megateriam and 
Burkholderia cepacia were the dominant bacterial species recovered from nematode- 
infected and uninfected roots. In each grove Bacillus megaterium was recovered from all 



62 

sites and Burkholderia cepacia was recovered from all but one site. Both species were 
more numerous on nematode-infected roots than on uninfected roots (Table 4-2). 
Arthrobacter ilicis, Stenotrophomonas maltophilia and Arcanobacterium haemolyticum 
also were recovered from both Tylenchulus-'mfected and uninfected roots in at least one 
site within each grove. The isolated fungal community was dominated by Fusarium 
solani in each of the three groves (Table 4-2). The 3-way analysis of variance showed no 
effect of nematode infection in roots on numbers of F. solani propagules; however a 
paired Mest of log-transformed data (pairing infected and uninfected root segments from 
each site within groves) indicated that nematode-infected roots contained more F. solani 
propagules than uninfected roots (df=14; t=3.44; P < 0.004). Species in the genera 
Trichoderma, Verticillum, Phythophthora, and Penicillum also were recovered from at 
least one sample from each of the groves. 

In vitro inhibition of P. nicotianae by the isolated bacteria. All isolated 
bacteria inhibited growth of P. nicotianae in vitro compared to the control treatment (Fig. 
4-1) (P < 0.05). There were no significant differences in degree of inhibition among the 
bacteria. 

Effect of nematode associated bacteria on the interaction between T. 
semipenetrans and P. nicotianae (laboratory study). Soil pH 4.5 was less favorable for 
seedling growth in the laboratory experiment compared to pH 7.0. Root and stem fresh 
weights in the untreated control treatment were 22% and 40% higher, respectively, at pH 
7.0 than at pH 4.5 (Figs. 4-2, 4-3). Higher pH also was more favorable for population 
growth of the bacteria, nematode, and fungus. Total bacteria CFUs were 75% more 
numerous at pH 7.0 than at pH 4.5 (Fig. 4-4). At pH 7.0 root infection by the nematode 



63 

was more than 4-fold (P < 0.05) that at pH 4.5. Mean ± standard error nematode female 
per gram root was 554.6 ± 1 10.9 at pH 7.0 and 121 ± 28.2 at pH 4.5. Corresponding 
means for offsprings per gram root were 2,583.9 ± 562.9 and 182.5 ± 24.1n plants 
infected only by P. nicotianae, fungal protein in roots was 72% greater at pH 7.0 than at 
pH 4.5 (P < 0.05). 

Phytophthora nicotianae was the only microorganism that reduced seedling root 
and stem fresh weights at either pH (Table 4-3; Figs 4-2, 4-3) (P < 0.001). Burkholderia 
cepacia had no significant effect on stem fresh weights but had a highly significant effect 
(P < 0.01) on root fresh weights which were 20% higher than those of controls. There 
were no interactions between this bacterium and P. nicotianae. Bacillus megaterium also 
increased stem (14%) and root (15%) fresh weights, and the bacterium interacted with P. 
nicotianae in this regard (P < 0.001). Further two-way ANOVA (pH and bacteria) 
demonstrated that B. megaterium had no effect on stem (P < 0.28) or root (P < 0.10) 
weight in the absence of P. nicotianae, but that the bacterium increased stem weight by 
55% (P < 0.002) and root weight by 80% (P < 0.001) in seedlings infected by the fungus 
compared to fungus only treatment. There were no interactions with pH in either 
analysis. With regard to P. nicotianae, two-way ANOVA (pH and fungus) showed that 
the fungus reduced the stem weights by 23% (P < 0.001) and root weight by 28% (P < 
0.001) in the absence of B. megaterium, but did not affect stem (P < 0.48) or root (P < 
0. 1 8) weights in the presence of the bacterium. The fungus effect did not interact with 
pH for these treatments. Despite the significant effect of B. megaterium and B. cepacia 
on the pathogenicity of P. nicotianae, neither bacterium affected the amount of fungal 
protein in the roots (Table 4-3; Fig. 4-5). 



64 

As noted previously, (Chapter 3;Table 3-1), the infection by T. semipenetrans 
increased growth of seedlings. Phytophthora nicotianae reduced stem and root fresh 
weight in the absence of T. semipenetrans but had no effect on stem and root weight in 
the presence of T. semipenetrans. There were interactions between T. semipenetrans and 
P. nicotianae with regard to root weight and amount of fungal protein in roots. 

Tylenchulus semipenetrans in combination with B. cepacia or B. megaterium at 
pH 7.0, reduced the fungal protein in the roots by 79% and 93% respectively, compared 
to the fungus only treatment (Fig. 4-5). At pH 4.5 the nematode in combination with 
either bacteria completely suppressed detection of P. nicotianae protein in roots. 
However, in general the combination of the nematode with either bacterium had few 
synergistic or additive effects on fungal development in roots or on fungal pathogenicity 
to seedlings. Three-way ANOVA (pH, nematode, bacterium) of treatments containing P. 
nicotianae showed that the nematode increased shoot and root weight (P < 0.001) and B. 
cepacia increased root weight (P < 0.007), but not shoot weight (P < 0. 19). There were 
no interactions between the nematode and bacterium (P < 0.87, and P < 0.97, 
respectively), suggesting an additive effect on root weight. The same analysis for B. 
megaterium showed that the bacterium increased shoot (P < 0.004) and root weights (P < 
0.001) of fungus-infected plants and demonstrated interactions between the bacterium 
and the nematode for both shoot (P < 0.006) and root weight (P < 0.002). Further two- 
way ANOVA (pH, nematode) revealed that the nematode increased shoot (P < 0.001) 
and root weights (P < 0.001) in the absence of the bacterium, but not in its presence (P < 
0.29, and P < 0.18, respectively). 



65 

In plants infected by P. nicotianae, the addition of T. semipenetrans increased the 
number of bacteria CFUs (P < 0.003) at both pH levels, regardless of inoculation with B. 
cepacia or B. megaterium (Fig. 4-4). An interaction occurred with pH for effect of the 
bacterium on the nematode population density. Burkholderia cepacia doubled numbers 
of nematode females and offspring (P < 0.001 and 0.004, respectively) in plants treated 
with T. semipenetrans and P. nicotianae at pH 4.5; however, the bacterium reduced (P < 
0.04) nematode offspring by 73% and did not affect females (P < 0.41) at pH 7.0. In 
plants infected by P. nicotianae and T. semipenetrans, B. megaterium increased numbers 
of female (320 vs 532; P < 0.01) and offspring (1,399 vs 1,690; P < 0.02) nematodes at 
both pH levels. 

Effect of nematode associated bacteria on the interaction between T. 
semipenetrans and P. nicotianae (greenhouse study). Burkholderia cepacia interacted 
with P. nicotianae in terms of stem and root weights (Table 4-4). Further one-way 
ANOVA showed that the fungus reduced the stem (36%; P < 0.003) and root weights 
(39%; P < 0.001) in the absence of B. cepacia, but did not affect stem (P < 0.75) or root 
(P < 0.1 1) weights in the presence of the bacterium. Similarly, B. cepacia increased stem 
(36%; P < 0.02) and root weights (39%; P < 0.03) in the presence of the fungus, but not 
in its absence {P < 0.95 and 0.31, respectively). In contrast to the laboratory experiment, 
B. megaterium had no effect on stem (P < 0.603) weight. However, results were 
consistent with the laboratory experiment for root weights, where B. megaterium 
interacted with P. nicotianae (P < 0.013). Further one-way ANOVA showed that the 
fungus reduced the root weight (38%; P < 0.001) in the absence of B. megaterium but did 
not affect the root weight (P < 0.23) in the presence of the bacterium. Similarly, B. 



66 

megaterium increased root weight by 39% (P < 0.06) in the presence of the fungus, but 
not in its absence (P < 0.1 1). As in the laboratory, neither B. cepacia nor B. megaterium 
affected the amount of P. nicotianae protein in roots (Table 4-4). 

The average (± standard error) nematode female per gram of root was 14.6 ± 3.6, 
and offspring per gram of root was 5.8 ± 3.01 . Although the nematode density was very 
low, the P. nicotianae protein in roots was 60% less in plants infected by both nematode 
and fungus than in plants infected by only the fungus (Fig. 4-5). Root and stem fresh 
weights were 39% and 25% greater (P < 0.05), respectively, in plants infected by both 
nematode and the fungus compared to plants infected by P. nicotianae alone (Figs. 4-2, 
4-3). There was interaction (P < 0.068) between T. semipenetrans and P. nicotianae with 
respect to root (P < 0.048) and stem (P < 0.06) fresh weights (Table 4-4). One-way 
ANOVA showed that stem and root fresh weights in fungal infected plants were 
increased by 23% (P < 0.05) and 28% (P < 0.0001), respectively, by the presence of T. 
semipenetrans. Phytophthora nicotianae protein in roots infected by both the fungus and 
nematode was less than half that in roots infected by only the fungus (Fig. 4-5), but in 
contrast to the laboratory experiment the effect was not significant (Table 4-4). 

In plants infected by P. nicotianae and B. cepacia, T. semipenetrans increased 
bacterial propagule numbers (Fig. 4-4) (P < 0.005). There was no effect of B. cepacia 
treatment on numbers of T. semipenetrans. Treatment with nematodes reduced the 
number of bacteria CFUs in seedlings treated with B. megaterium and P. nicotianae (P < 
0.06). In contrast to the laboratory experiment, B. megaterium reduced (P < 0.046) 
nematode offspring in plants treated with P. nicotianae and T. semipenetrans but did not 
affect the number of nematode females. 



67 

Discussion 

Tylenchulus semipenetrans altered the microbial community in the citrus 
rhizosphere by increasing propagule densities of bacteria and fungi in each of the three 
groves studied. All of the isolated bacteria suppressed growth of P. nicotianae in vitro; 
however, in contrast to T. semipenetrans, no bacteria inhibited growth of the fungus when 
inoculated in whole-plant experiments. Nevertheless, the nematode and both of the 
selected bacteria increased the growth of citrus seedlings infected by P. nicotianae. 
These results suggest that multiple mechanisms may attenuate of virulence of P. 
nicotianae in roots infected by T. semipentrans. 

The positive effect of T. semipentrans and P. nicotianae on population increase of 
rhizosphere microorganisms likely results from leakage of nutrients from fungus-induced 
root lesions, nematode infection sites, or gelatinous egg masses. Our results agree with 
Weller, (1986) who reported that densites of indigenous, gram-negative bacteria of 
Pseudomonas spp. were greater on roots infected by Gaeumannomyces graminis var 
tritici than on healthy plants. Rovira and Wildermuth (1981) used electron microscopy to 
show that bacteria proliferate in fungus-induced lesions. Bergeson et al., (1972) showed 
a significant increase of Fusarium propagules in the rhizosphere of roots inoculated 
simultaneously with M. javanica and F. oxysporium f. sp. lycopersici compared to roots 
inoculated with only the fungus. 

The dominant bacterial species isolated in these surveys, B. megaterium and B. 
cepacia, are well documented biological control agents (Liu and Sinclair, 1993; Schroth 
and Hancock, 1981; Mao et al., 1998; Zheng and Sinclair, 1996). Both species have been 
described as " plant growth promoting rhizobacteria" (PGPR; Schroth and Hancock, 
1981) because of their ability to improve plant growth by aggressively colonizing roots 



68 

and preempting the establishment of deleterious rhizosphere microorganisms (Suslow et 
al., 1982). Bacillus megaterium forms endospores that are resistant to unfavorable 
conditions. The bacterium is a good root colonizer, rhizosphere competitor, and remains 
viable for extended periods (Liu and Sinclair, 1993). Multiple effects of B. megaterium 
on soil microorganisms have been documented (Liu and Sinclair, 1993) and the 
bacterium has been shown to be a potential biocontrol agent for Rhizoctonia root rot of 
soybeans (Zheng and Sinclair, 1 996). B. cepacia has been reported to colonize and 
enhance root hair development (De Freitas and Germida, 1990) and to produce wide 
spectrum antifungal metabolites (Lambert et al., 1987). The bacterium has been studied 
for biological control of diseases caused by many plant pathogenic fungi on different 
crops such as R. solani, Pythium ultimum (Mao et al., 1998) and F. oxysporum (Larkin- 
Robert and Fravel-Deborah, 1998) on tomato, and damping-off diseases caused by 
species of Pythium and Fusarium (Mao, et al., 1997, 1998) on corn seedlings and F. 
oxysporium f sp. cepae on onion seedlings (Kawamoto and Lorbeer, 1976). PGPR also 
can alter plant physiology and increase the host plant defenses to pathogen attack 
(induced resistance). Colonization of roots by PGPR suppressed anthracnose of 
cucumber leaves caused by Colletotrichum orbicular (Wei et al, 1991). Root 
bacterization with Pseudomonas strain WC 5417r reduced the incidence of fusarium wilt 
in carnation and the amount of F. oxysporium f. sp. dianthi in stems of the plants (Van 
Peerretal., 1991). 

The suppressive effect of B. cepacia and B. megaterium on growth of P. 
nicotianae in vitro is in agreement with Turney et al. (1992) who noted that all bacterial 
isolates collected from citrus rhizosphere soil inhibited growth of P. nicotianae on agar 



69 

plates. However, no general relationship exists between the ability of a bacterium to 
inhibit a pathogen in vitro and suppress disease caused by the pathogen in vivo (Baker, 
1987; Schroth and Hancock, 1981). Despite their potential as biological control agents, 
neither bacteria in this study showed evidence of suppressing the rate of infection or 
growth of P. nicotianae in citrus seedlings. Therefore, the results of the whole plant 
experiments suggest that these bacteria may increase tolerance of citrus seedlings to 
infection by P. nicotianae. Both bacteria increased weights of citrus seedlings infected 
by P. nicotianae, and in general the fungus did not significantly affect weights of the 
roots or stems of seedlings that were treated with either bacteria. We did not investigate 
causes of bacteria-induced seedling tolerance to P. nicotianae. Possible mechanisms by 
which PGPR can enhance plant tolerance are poorly understood, but may involve 
favorable modifications to the rhizosphere chemistry. Bacillus megaterium and other 
Bacillus spp. formulated commercially as Phosphobacterin (Copper, 1959; Mishustin, 
1963) increased the vigor of wheat in the greenhouse but not in the field, possibly by 
transformation of unavailable minerals and organic compounds or by production of 
biologically active substrates such as auxins or gibberellins (Broadbent et al., 1977; Burr 
etal., 1978). 

The effect of T. semipenetrans on growth of P. nicotianae-inkcted seedlings in 
the greenhouse was consistent with those in the laboratory experiment which were 
reported previously (El-Borai et al., 2000). The general lack of additive or synergistic 
effects by combinations of bacteria and nematodes is not surprising since treatment with 
either bacteria or nematodes tended to result in normal seedling growth similar to that of 
untreated controls. Moreover, these experiments were not controlled to the extent that 






70 

involvement by these two bacteria in the effects demonstrated by the nematode treatment 
can be discounted. All treatments with T. semipenetrans increased microbial populations 
and both bacteria were encountered in all treatments by the end of the experiment. 
However, unlike either bacterium, all treatments with nematodes reduced the amount of 
P. nicotianae protein detected in roots. This is the only direct evidence from these 
experiments for a possible mechanism by which pathogenicity of the fungus is attenuated 
by the presence of another organism. Eggs of T. semipenetrans were shown recently to 
be inhibitory to the growth of P. nicotianae (El-Borai Kora et al., 2001). Therefore, there 
is a good likelihood that infection of citrus roots by T. semipenetrans reduces population 
growth off. nicotianae by direct antibiosis, which mitigates virulence of the fungus. All 
T. semipenetrans-induced changes in the microbial community revealed in this study 
appear to be favorable to the citrus root system; however, whether augmentation of 
natural levels of bacteria by nematodes has a significant effect on the plant is unknown. 

The effects of B. cepacia on T. semipenetrans offspring at higher pH were 
consistent with Meyer et al. (2001) who showed that B. cepacia (Bc-2 and Bc-F) and 
Trichoderma virens (Gl-3) significantly suppressed numbers of root-knot nematode eggs 
and juveniles on roots of pepper plants. Psudomonas aureofaens inhibited Criconemella 
xenoplax egg hatch in vitro and reduced nematode population densities in the greenhouse 
(Westcott and Kluepfel, 1993). The effect of B. megaterium on T. semipenetrans in the 
greenhouse experiment was consistent with Neipp and Becker (1999) who showed that 
two strains of B. megaterium inhibited hatching Heterodera schachtii from cysts in vitro 
and reduced nematode numbers and infection of sugarbeet seedlings when eggs were 
used as inoculum. However the bacterium did not affect H. schachtii root infection in 



71 

growth pouches. Treating potato plants with B. megeaterium reduced population 
densities of Meloidogyne chitwoodi and Pratylenchus neglectus by up to 50% (Al- 
Rehiayani et al., 1999). Nevertheless, both bacteria increased numbers of T. 
semipenetrans under some of our experimental conditions. Differences in nematode 
species, or concomitant infection of the nematode with P. nicotianae, may account for 
variable effects of the bacteria on T. semipenetrans and other nematode species. 

In conclusion, these studies indicate that infection of citrus roots by T. 
semipenetrans increases population densities of rhizosphere microorganisms, some of 
which may increase the tolerance of citrus seedlings to infection by P. nicotianae. 
However, we found no evidence that reduced infection of roots by the fungus in the 
presence of T. semipenetrans is mediated by changes in the rhizosphere microbial 
community. Additional studies that better control the combinations of nematodes, fungi, 
and bacteria, by excluding background contamination, are needed to demonstrate a direct 
effect of the nematode on suppression of root infection by the fungus. Field studies are 
also warranted to determine if disease caused by P. nicotianae is reduced by the 
nematode and whether the effect is economically important. Nevertheless, this study 
provides further evidence that T. semipenetrans is unlikely to exacerbate fibrous root rot 
of citrus caused by P. nicotianae (El-Borai Kora et al., 2001). 



72 



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Bacteria species 

Figure 4-1. Effect of nematode-associated bacteria on Phytophthora nicotianae 
mycelial growth in vitro. Bars followed by a common letter are not 
significantly different according to Dunnett test (P < 0.05). B. m= 
Bacillus megaterium, B. c=Burkholderia cepacia, A. h= 
Arcanobacterium haemolyticum, A. i=Arthrobacter ilicis, 
S. m-Stenotrophomonas maltophilia. 






75 






0.5 

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I 



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3 



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I Bacteria+fungus+nematode 



X 



1 









I 



X 



Burkholderia 
cepacia 



Bacillus 
megaterium 



laboratory exp. 
(pH=7.0) 



laboratory exp. 
(pH=4.5) 




greenhouse exp. 
(pH=4.5) 




N F N C 



Figure 4-2. Effect of Burkholderia cepacia, Bacillus megaterium and Tylenchulus 
semipenetrans on citrus seedlings growth and virulence of Phytophthora 
nicotianae to seedlings root fresh weight. Bars indicate the standard error 
of the mean for eight seedlings replications per treatment. N=Nematode, 
F=Fungus, NF=Nematode+Fungus, and C=Untreated control. 









76 






'53 

CO 

B 

e 

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00 




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0.4 



] Bacteria alone 
bacteria+fungus 
Bacteria+fungus+nematode 



laboratory exp. 
(pH=4.5) 




Burkholderia 
cepacia 



Bacillus 
megaterium 



N F N C 



Figure 4-3. Effect of Burkholderia cepacia, Bacillus megaterium, and Tylenchulus 
semipenetrans on citrus seedlings growth and virulence of Phytophthora 
nicotianae to seedlings stem fresh weight. Bars indicate the standard error 
of the mean for eight seedlings replications per treatment. N=Nematode, 
F=Fungus, NF=Nematode+Fungus, and C=Untreated control. 



77 



Table 4-3. Analyses of variance of effects of Burkholderia cepacia, and Bacillus 
megaterium.on stem fresh weight, root fresh weight, and Phytophthora 
nicotianae protein in citrus roots in the laboratory. 







Stem 


weight 


Root 


weight 


Fungal 


protein 




F-value 


P-value 


F-value 


P-value 


F-value 


P-value 










Burkholderia cepacia 






pH 




33.20 


0.000 


0.90 


0.347 


4.27 


0.049 


Fungus 




8.60 


0.005 


12.60 


0.001 


20.80 


0.000 


Bacteria 




0.16 


0.693 


6.12 


0.016 


1.50 


0.230 


pH x Fungus 




0.90 


0.346 


1.19 


0.281 


2.36 


0.136 


pH x Bacteria 




1.15 


0.287 


0.60 


0.443 


1.17 


0.290 


Fungus x Bacteria 


0.94 


0.337 


0.74 


0.392 


0.16 


0.697 


pH x Fungus x 


Bacteria 


0.51 


0.478 


1.18 


0.283 


0.06 


0.802 










Bacillus megaterium 






pH 




37.50 


0.000 


5.72 


0.020 


2.40 


0.134 


Fungus 




0.76 


0.388 


0.83 


0.367 


25.80 


0.000 


Bacteria 




11.90 


0.001 


28.90 


0.000 


0.35 


0.557 


pH x Fungus 




3.18 


0.080 


1.28 


0.262 


2.75 


0.110 


pH x Bacteria 




0.83 


0.366 


0.63 


0.430 


0.23 


0.639 


Fungus x Bacteria 


4.42 


0.040 


10.10 


0.002 


0.30 


0.589 


pH x Fungus x 


Bacteria 


0.26 


0.609 


0.78 


0.382 


0.07 


0.796 



78 



Table 4-4. Analyses of variance of effects of Burkholderia cepacia, Bacillus 

megaterium, and Tylenchulus semipenetrans on stem fresh weight, root fresh 
weight and Phytophthora nicotianae protein in citrus roots in the greenhouse. 





Stem 


weight 


Root 


weight 


Fungal 


protein 




F-value 


P-value 


F-value 


P-value 


F-value 


P-value 








Burkholderia cepacia 






Fungus 


5.51 


0.230 


22.90 


0.001 


22.25 


0.001 


Bacteria 


3.76 


0.059 


1.04 


0.314 


0.46 


0.504 


Fungus x Bacteria 


3.44 


0.070 


5.76 


0.021 


2.09 


0.159 








Bacillus megaterium 






Fungus 


14.06 


0.001 


19.50 


0.001 


9.09 


0.005 


Bacteria 


0.27 


0.603 


0.26 


0.614 


2.66 


0.114 


Fungus x Bacteria 


0.39 


0.536 


6.81 


0.013 


0.03 


0.860 








Tylenchulus semipenetrans 






Nematode 


1.70 


0.199 


3.15 


0.082 


2.16 


0.155 


Fungus 


16.38 


0.002 


36.90 


0.001 


11.27 


0.003 


Nematode x Fungus 


3.48 


0.068 


4.10 


0.048 


2.25 


0.146 



79 



p 
U 

■c 

i 



1000 - 


laboratory exp. 
(pH=7.0) 


750 


T 


i 


500 - 


T 




250 

n 


_._ 


1 T 


■ ■ll 


1000 - 


I 1 Bacteria alone laboratory exp. 

i 1 bacteria+fungus (pH=4.5) 

mmmm Bacteria+fungus+nematode 


750 - 




500 - 




250 
n - 


,^ 


1 nl^ll --■ 


u 
2250 - 


-r- 


greenhouse exp. 
(pH=4.5) 


1500 - 




I 


750 




1 l.l 





T 


Fi 


n* III. 



Burkholderia Bacillus 
cepacia megaterium 



N F N C 
F 



Figure 4-4. Numbers of bacteria colony forming units recovered from citrus seedlings. 
Bars indicate the standard error of the mean for five seedlings replications 
per treatment. N=Nematode, F=Fungus, NF=Nematode+Fungus and 
C=Untreated control. 



80 



¥ 



o 

•8 



c 
'5 

a 

ex 






2.0 
1.5 
1.0 
0.5 

0.0 

2.0 

1.5 
1.0 



73 0.5 



0.0 
2.0 

1.5 

1.0 

0.5 ] 

0.0 



I 



J L 



r*n 



I I Bacteria alone 

I . I Bacteria+fungus 

l l bactcria+fungus+ncmatode 



I 



laboratory exp. 
(pH=7.0) 



laboratory exp. 
(pH=4.5) 




T greenhouse exp. 
(pH=4.5) 




Burkholderia Bacillus 

cepacia megaterium 



N F N C 



Figure 4-5. Effect of Burkholderia cepacia. Bacillus megaterium, and Tylenchulus 
semipenetrans on absorbance of Phytophthora nicotianae protein in 
citrus roots (measured by ELISA test). N=Nematode, F=Fungus, 
NF=Nematode+Fungus and C=Untreated control. 



CHAPTER 5 

EGGS OF TYLENCHULUS SEMIPENETRANS INHIBIT GROWTH OF 
PHYTOPHTHORA NICOTIAN AE AND FUSARIUM SOLANI IN VITRO 

Introduction 

Citrus nematode, Tylenchulus semipenetrans Cobb, is a semi-endoparasite of the 
cortical cells of citrus fibrous roots. The anterior portion of the female extends several 
cell layers deep in the cortical parenchyma, while the posterior portion, outside of the 
root, secretes a gelatinous matrix into which eggs are deposited (Van Gundy, 1958). The 
eggs with this protective gelatinous matrix is known as an egg-mass (Maggenti, 1962). 
Egg masses contain up to 75-100 eggs (Baines, 1950). The female of T. semipenetrans is 
sessile, obtaining its nutrients from specialized transfer cells (6 to 10) called "nurse" cells 
around the nematode head (Van Gundy, 1958). These "nurse" cells are required for 
successful reproduction and die upon the female's death. 

Interactions between the citrus nematode T. semipenetrans and fibrous root rot 
fungus Phytophthora nicotianae Dastur Breda de Haan (synonym = parasitica) (Hall, 
1993) in citrus have been shown to be antagonistic to the fungus (El-Borai et al., 2000). 
Citrus seedlings infected by the nematode and later inoculated with the fungus, grew 
larger and contained less fungal protein in the root tissues than plants not infected by the 
nematode. Both organisms feed in the cortex and have been shown to reduce the mass of 
the fibrous root system (Duncan et al., 1993) and impact citrus yield (Duncan et al., 1993; 
Graham and Menge, 1999). 



81 



82 

Interactions involving nematodes and fungi have been studied extensively and are 
often synergistic (Atkinson, 1892; Bergeson, 1972; Carter, 1981; MacGuidwin and 
Rouse, 1990; McLean and Lawrence, 1993a, 1993b & 1995; Mai and Abawi, 1987; 
Powell, 1971a, 1971b; Powell et al., 1971; Powelson and Rowe, 1993; Prot, 1993; Roy et 
al., 1989; Whitney, 1974; Webster, 1985). Only occasionally are the interactions 
antagonistic (Valle-Lamboy and Ayala, 1980., Costa Manso and Huang, 1986., Gray et 
al., 1990., Sankaralingam and McGawely, 1994). Orion and Kritzman (1991) and Papert 
and Kok (1999) reported that the gelatinous matrix of the root-knot nematode, 
Meloidogyne javanica and Meloidogyne fallax provide protection against microbial 
attack either due to antibiotic compounds from the matrix or due to associated bacteria. 

Possible mechanisms by which the citrus nematode suppresses fungal development 
include direct chemical antagonism by the nematode, nutrient competition, or alteration 
of the microbial community in the rhizosphere to favor microorganisms antagonistic to P. 
nicotianae. The objective of this study was to determine the effect of T. semipenetrans 
eggs on P. nicotianae and Fusarium solani in vitro compared to eggs of the root-knot 
nematode M. arenaria. 

Materials and Methods 

Bioassays were conducted to determine the effect of eggs of two nematodes, T. 
semipenetrans and M. arenaria on P. nicotianae and F. solani mycelial growth in vitro in 
three different experiments. In the first experiment, Tylenchulus semipenetrans inoculum 
was obtained from naturally-infected roots from the field. Eggs, juveniles and males 
were scrubbed from root surfaces and collected on 74/ 25 urn pore nested sieves. 
Nematodes were further separated from soil and plant debris by sucrose centrifugal- 



83 

flotation method (Jenkins, 1964) followed by magnesium sulfate fractionation. The 
magnesium sulfate (225.9g/liter water) solution was underlayed beneath nematode 
suspensions, then centrifuged for three minutes at 1,500 rpm (Hendrickx et al., 1976). 
The interface containing the nematodes was drawn off using a 5-mL pipet. Nematodes 
were rinsed repeatedly with tap water over a 635 urn pore sieve to remove residual 
magnesium sulfate. To separate eggs from vermiform stages, a 325 [im pore sieve was 
used to retain the vermiform stages (as well as free living nematodes). Approximately 15 
to 20 passes of the nematode suspension in greater than 1,000 volumes of water was 
sufficient to purify eggs. The egg suspension was then concentrated on a 20-um-pore 
sieve. The few remaining free-living nematodes in the suspension were hand picked. 

In the second experiment, eggs of T. semipenetrans were scrubbed from root 
surfaces using 5% commercial bleach (0.03% NaOCl) for 30 seconds. Bleach was used 
to ensure that no residue of the matrix remained on eggs. 

In the Third experiment, an isolate of M. arenaria (Neal) Chitwood race 1 from 
Levy County, Florida was used. This isolate was cultured on tomato (Lycopersicon 
esculentum Mill. cv. Rutgers) in steam-pasteurized potting soil. The eggs were extracted 
either by scrubbing the galled tomato roots gently in 5% commercial bleach (0.03% 
NaOCl) for 30 seconds (Hussey and Barker, 1973; McClure et al., 1973) and caught on a 
25 urn pore sieve) or with the same procedure but without using bleach. The same 
magnesium sulfate and sieving procedures described for T. semipenetrans were used to 
remove debris and separate the M. arenaria eggs from juveniles and other nematodes. 

Eggs in all experiments were surface-sterilized in a laminar flow hood. Nematode 
eggs were back-washed into 12-ml sterile disposable plastic tubes. The egg suspension 



84 

was allowed to settle for 1 hour and volume was reduced to 0.5 ml of egg suspension per 
tube using a sterilized 5-ml pipet. Three treatments were used in each experiment 
conducted: live-surface sterilized eggs, heat-killed surface-sterilized eggs (60 °C for 10 
minutes), and a water control. Both live and heat-killed surface sterilized eggs were 
treated with cupric sulfate (0.1%) for 30 minutes; mercuric chloride (0.025%) for 10 
minutes; and then streptomycin sulfate (0.2%) for 24 hours. The eggs were rinsed seven 
times on an autoclaved 25-um-pore sieve with 1 -liter exchanges of sterile distilled water 
between each sterilant. 

Approximately, 35,000 eggs of each nematode in a 5-p.L water droplet were 
deposited in the center of nutrient agar in 100 x 15 mm Petri plates. Nutrient agar plugs 
(5-mm diam.) cut with a cork borer from margins of actively growing colonies of either 
P. nicotianae or F. solani fungal isolates were placed on the agar surface over the eggs 
and incubated at room temperature. Control plates received 5-pL sterile distilled water in 
place of nematode eggs. After 48 hours, fungal colony growth diameter was determined 
by means of linear measurements made in eight directions starting from the center of the 
fungal plug using a template made from an inverted Petri dish placed under the dish and 
measurements averaged. Each treatment was replicated 15 times (one plate per 
replicate). 

To independently test the effects of the sterilization chemicals on fungal growth, a 
series of dilutions (0, 10°, 10" 2 , 10" 3 , and 10" 4 ) were made of the original concentrations of 
each compound used to surface-sterilize the eggs. Five pL from each dilution were 
pipetted onto the center of nutrient agar, and a plug of P. nicotianae was then placed on 
top. After 48 hours, P. nicotianae colony growth diameter was measured as described 



85 

previously. Eight plates were used with each dilution, for each chemical sterilant. Both 
live and heat-killed eggs of both nematode species were observed for hatching in a 60 x 
15-mm Petri plate for a 1 -month period. Approximately 1,000 eggs of each nematode 
species from each treatment in 3-ml sterile distilled water were counted weekly. 

All experiments were conducted twice. Data were analyzed by one-way Analysis 
of Variance (ANOVA), and mean separation was determined with Duncan's multiple 
range test at (P <0.05). 

Results 

Surface-sterilized live T. semipenetrans eggs inhibited P. nicotianae mycelial 
growth by 74% compared to water controls and 72% compared to heat-killed eggs (Figs. 
5-1 A; 5-2 A) (P <0.05). In a repeated experiment (Fig. 5- IB) with the same treatments, 
live as well as heat-killed surface-sterilized eggs were inhibitory and both reduced P. 
nicotianae colony growth by 94% in both treatments compared to controls. Tylenchulus 
semipenetrans eggs extracted with bleach did not differ in their activity against P. 
nicotianae mycelial growth compared to eggs extracted without bleach (Fig. 5-1C). Live 
surface-sterilized T. semipenetrans eggs extracted without bleach inhibited F. solani 
mycelial growth by 92% compared to controls and heat-killed eggs (Figs. 5-3A; 5-2B). 
In the repeated experiment, both the live and heat-killed surface-sterilized eggs of T. 
semipenetrans inhibited F. solani mycelial growth and reduced colony diameter by 95% 
compared to water controls and 55% compared to heat-killed eggs (Fig. 5-3B; P <0.05). 
With bleach extraction, both live and heat-killed eggs inhibited F. solani colony diameter 
by 94% (P <0.05) compared to water controls after 36 hours (Fig. 5-3C). However, 
heat-killed eggs lost some of their inhibitory activity compared to live eggs after 72 



86 

hours. Live T. semipenetrans eggs inhibited F. solani colony growth by 79% compared 
to 55% inhibition from the heat-killed eggs (Fig. 5-3C).Live M. arenaria eggs had no 
comparable effect on P. nicotianae mycelial growth (Fig. 5-5 A) the effect of M. arenaria 
eggs on P. nicotianae was the same with or without bleach extraction (Fig. 5-4A). The 
effect of M. arenaria on F. solani mycelial growth contrasted with the effect on P. 
nicotianae. Live M. arenaria surface-sterilized eggs extracted with bleach did not inhibit 
F. solani mycelial growth (Figs. 5-4B; 5-5B) compared to controls; but without bleach, 
the mycelial growth of F. solani was inhibited by 64% compared to water controls (Fig. 
5-4B). 

The original concentrations of 0.025% mercuric chloride and 0.2% streptomycin 
sulfate, each individually inhibited P. nicotianae mycelial growth by 100% and 66%, 
respectively, compared to water controls. However, there were no significant differences 
in mycelial growth with any other dilutions of either sterilant compared to water controls 
(Table 5-1). Cupric sulfate had no significant effect on the mycelial growth of P. 
nicotianae. When all three surface sterilants were mixed together, the original 
concentration was the only treatment that had a significant inhibitory effect on P. 
nicotianae colony growth (100%) compared to all other dilutions and water controls 
(Table 5-1) (P<0.05). 

Sixty-five percent of surface-sterilized T. semipenetrans eggs hatched compared to 
75% of eggs rinsed with water only. Seventy-five percent of surface-sterilized M. 
arenaria eggs hatched compared to 90% of eggs rinsed with water only. No juveniles 
hatched from heat-killed eggs in any experiment. 



87 

Discussion 

The results of these experiments demonstrated a direct, species-specific effect of 
citrus nematode eggs on P. nicotianae and F. solani mycelial growth. Though the heat- 
killed eggs of T. semipenetrans in the repeated experiment showed an inhibitory effect on 
P. nicotianae and F. solani, the more consistent inhibition of live eggs suggests that eggs 
may actively secrete compounds that inhibit fungal growth. It is unlikely that the 
disinfectants used to surface-sterilize the eggs caused these effects since a one hundred- 
fold dilution of the original concentration of these compounds was sufficient to alleviate 
any fungal inhibition. Eggs were rinsed seven times with 1-L exchanges of sterile 
distilled water between each sterilant, to demonstrate that the inhibition effect on P. 
nicotianae was due to a direct effect of the eggs and not the disinfectants. 

We showed previously that the interaction between T. semipenetrans and P. 
nicotianae is antagonistic to the fungus (El-Borai et al., 2000). Tylenchulus 
semipenetrans interfered with P. nicotianae, reducing levels of infection in roots and 
producing increased growth of citrus seedlings compared with seedlings infected by P. 
nicotianae alone. This study suggests that T. semipenetrans eggs secrete antifungal 
compounds that inhibit the development of P. nicotianae in roots. Similarly, the 
gelatinous matrix of root-knot nematode has been shown to provide protection against 
microbial attack (Orion and Kritzman, 1991; Papert and Kok, 1999). However, this is the 
first study to demonstrate inhibition of fungal mycelial growth by nematode eggs. 
Inhibition by T. semipenetrans eggs of P. nicotianae and F. solani with or without bleach 
extraction suggests that the effect is likely due to chemicals secreted by eggs and is not 
contained in the gelatinous matrix which is dissolved and removed by the bleach. 
Conversely, M. arenaria eggs extracted without bleach did inhibit F. solani mycelial 



88 

growth whereas those extracted with bleach did not, suggesting that the residual 
gelatinous matrix may contain constitutive compounds that inhibit the growth of certain 
fungi. Differences in the effects of eggs of root-knot and citrus nematodes on plant 
pathogenic fungi may result from adaptation to different parasitic behaviors. Because the 
female root knot nematode remains sessile inside the developing root tissue for most of 
its life (Bird, 1962), this species may experience less selection pressure to protect its 
feeding site compared to the semi-endoparasitic T. semipenetrans that infects and remains 
exposed on portions of the roots that already fully developed (Conn, 1965). 

Nematode fungal interactions are often synergistic. There are few examples of 
antagonistic interactions between nematodes and fungi. Gray et al. (1990) found that 
survival of alfalfa seedlings was lower following a single inoculation with only 
Phytophthora megasperma f. sp medicaginis than following inoculation with both 
Meloidogyne hapla and P. megasperma f. sp medicaginis. The root-knot nematodes 
Meloidogyne incognita have been shown to interfere with the development of the fungus 
Pythium graminicola on roots of sugarcane (Valle-Lamboy and Ayala, 1980). The 
presence of the nematode in combination with the fungus interfered with the fungus 
development on roots, and the fungus partially reduced the detrimental effect of the 
nematode. The antagonistic interaction between both organisms was beneficial to the 
plants which grew and developed better when both were together than when the two 
microorganisms act separately. Also, M. incognita has shown to protect Phaseolus 
vulgaris roots from the fungus Rhizoctonia solani (Costa Manso and Huang, 1986). 
Sankaralingam and McGawely (1994) also reported an antagonistic interaction between 
the reniform nematode Rotylenchulus reniformis and the cotton seedling blight fungus 



89 

Rhizoctonia solani. The combined effect of the nematode and the fungus was 
antagonistic, with respect to cotton seedling blight. Because T. semipenetrans and R. 
reniformis are both sedentary semi-endoparasites with similar life histories, it would be 
interesting to test activity of eggs of/?, reniformis for activity against other plant 
pathogenic fungi. 

Tylenchulus semipenetrans eggs had an antagonistic effect on P. nicotianae 
mycelial growth in vitro. These results may explain T. semipenetrans-medizted 
suppression of root infection by P. nicotianae that resulted in increased citrus seedling 
growth relative to seedlings infected with the fungus alone (El-Borai et al., 2000). 
Further work is warranted to identify and characterize the compounds secreted by eggs of 
T. semipenetrans that inhibit the growth of P. nicotianae. 



90 






2 
•3 
>> 
a 

_o 

Q 

o 

« 

E 

a 
•B 

8 



1 

i 

I 



100 

80 

60 

40 

20 

80 
60 -\ 
40 
20 


80 
60 ^ 
40 
20 




B 



a 




b 
00 



T. semipenetrans 
no bleach 



T. semipenetrans 
no bleach 




T. semipenetrans 
with bleach 



b 
00 



Water 


Live surface 


Heat-killed 


control 


sterilized 


surface sterilized 




eggs 


eggs 



Figure 5-1 . Effect of Tylenchulus semipenetrans eggs on Phytophthora nicotianae 
mycelial growth after 48 hour in vitro. A, B and C are the results 
of the three experiments. Bars indicate the standard error of the mean for 
15 replications per treatment. Bars followed by a common letter are not 
different according to Duncan's multiple range test (P > 0.05). 



91 



Tylenchulus semipenetrans 



Phytophthora nicotianae 

A 



Fusarium solani 



B 




l-Water control 

P. nicotianae 

only 



2-Live surface 
sterilized eggs 



r i 



3-Heat-killed surface 
sterilized eggs 




Figure 5-2. Effect of Tylenchulus semipenetrans eggs on Phytophthora nicotiane and 
Fusarium solani mycelial growth after 48 hr in vitro. Eggs were extracted 
without bleach. Al Bl= Water control (Phytophthora nicotianae only), 
A2B2 =Tylenchulus semipenetrans live surface sterilized eggs, A3B3= 
Tylenchulus semipenetrans heat-killed surface sterilized eggs. 



92 



-t— » 



•a 

o 
o 

s 

.3 

o 



I 



r. semipenetrans 
no bleach 




80 - 



200 



150 

100 

50 





T. semipenetrans 
no bleach 





36 hour 
72 hour 



T. semipenetrans 
with bleach 



B 






Water 
control 



Heat-killed surface 
sterilized 



Live surface 
sterilized 

eggs eggs 

Figure 5-3. Effect of Tylenchulus semipenetrans eggs on Fusarium solani mycelial growth 
after 48 hr in A+B and 36 +72 hour in C in vitro . A and B are the results of 
two replicate experiment. Bars indicate the standard error of the mean for 15 
replications per treatment. Bars followed by a common letter are not different 
according to Duncan's multiple-range test (P > 0.05). 



93 



Meloidogyne arenaria 



80 i 



60 - 



40 



20 






*3 

o 
U 



- 



100 - 



80 



60 



40 



20 - 







Water control 

Live surface sterilized eggs 

Dead surface sterilized eggs 



Phytophthora nicotianae 







Fusarium solani 



B 




With bleach 



Without bleach 



Figure 5-4. Effect of Meloidogyne arenaria eggs (extracted with and without bleach) 
on Phytophthora nicotianae (A) and Fusarium solani (B) mycelial growth 
after 48 hour in vitro. Bars indicate the standard error of the mean for 15 
replications per treatment. Bars followed by a common letter within each 
experiment are not different according to Duncan's multiple range test at 
(P<0.05). 



94 



Meloidogyne arenaria 

Phytophthora nicotianae 



Fusarium solani 



B 




l-Water control 

P. nicotianae 

only 



2-Live surface 
sterilized eggs 



3-Heat-killed surface 
sterilized eggs 




Figure 5-5. Effect of Meloidogyne arenaria eggs on Phytophthora nicotianae and 

Fusarium solani mycelial growth after 48 hr in vitro. Eggs were extracted 
with bleach. AlBl=Water control treatment (Phytophthora nicotianae only, 
A2B2=Meloidogyne arenaria live surface sterilized eggs, A3B3= 
Meloidogyne arenaria heat-killed surface sterilized eggs. 



95 



Table 5-1. Effect of cupric sulfate, mercuric chloride and streptomycin sulfate on 
Phytophthora nicotianae mycelial growth after 48 hours in vitro. 







Phytophthora 


nicotianae colony 


diameter (/im) 










Cupric sulfate 




Cupric 


Mercuric 


Streptomycin 


Mercuric chloride 


Dilutions 


sulfate 


chloride 


sulfate 


Streptomycin sulfate 


00 


57.79 a 


57.79 a 


57.79 a 


22.36 a 


10° 


59.04 a 


29.00 b 


39.40 b 


0.00 b 


io- 2 


59.79 a 


55.92 a 


58.02 a 


23.11 a 


io- 3 


60.77 a 


57.77 a 


56.17 a 


23.13 a 


IO" 4 


63.75 a 


59.02 a 


58.71 a 


23.38 a 



Numbers are the means of eight replications for each dilution. Numbers in a column 
followed by a common letter are not significantly different according to Duncan's multiple range 
test (P < 0.05). 



CHAPTER 6 
RESEARCH SUMMARY AND CONCLUSIONS 

Plant parasitic nematodes can play a major role in disease interactions involving a 
variety of other organisms. Interactions involving nematodes often have important 
economic effects on the survival and growth of plants and they contribute substantially to 
variability in crop growth. The most common association between plant parasitic 
nematodes and fungi in citrus is that between citrus nematode Tylenchulus semipenetrans 
and fibrous root rot fungus Phytophthora nicotianae. Both parasites occupy the fibrous 
root cortex and both are economically important pathogens. There are few data reporting 
the influence of these two pathogens on one another; however, a recent study found that 
chemical suppression of citrus nematode was related to increased density of P. nicotianae 
propagules in citrus orchards. The present research was initiated to test whether the 
nematode does in fact suppress P. nicotianae and to investigate the mechanism(s) by 
which such an interaction might occur. 

A series of in vitro bioassays and whole plant experiments were conducted to 
investigate the nature of the interaction between both organisms. A bioassay revealed 
that fibrous root segments containing T. semipenetrans females and egg masses were 
subsequently infected by P. nicotianae only half as often as were uninfected root 
segments. In whole plant experiments conducted in the laboratory and greenhouse, citrus 
seedlings infected by the nematode and later inoculated with the fungus grew larger and 
contained less fungal protein in the root tissues than did plants infected by only the 



96 



97 

fungus. Results of this series of experiments demonstrated a significant interaction 
between T. semipenetrans and P. nicotianae in which the nematode is antagonistic to the 
fungus. 

Possible mechanisms by which the nematode might inhibit root infection by the 
fungus include direct antibiosis, resource competition, and indirect mediation by 
microorganisms associated with nematode feeding sites. Following the later hypothesis, 
a field survey was initiated to investigate whether T. semipenetrans alters the 
composition of rhizosphere inhabiting microorganisms, and to determine whether 
microorganisms associated with the nematode affect the behavior of P. nicotianae. In 
each of three citrus orchards the numbers of bacteria colony forming units from T. 
semipenetrans -infected roots were greater than those from uninfected roots. Bacillus 
megaterium and Burkholderia cepacia were the dominant bacterial species recovered and 
both were more numerous on nematode-infected roots than on uninfected roots. 
Arthrobacter ilicis, Stenotrophomonas maltophilia and Arcanobacterium haemolyticum 
were commonly encountered at lower propagule density. The fungal community was 
dominated by Fusarium solani, but Trichoderma, Verticillum, Phythophthora, and 
Penicillum were also recovered. All isolated bacteria inhibited growth of P. nicotianae in 
vitro. Experiments were then conducted using combinations of selected bacteria 
{Bacillus megaterium and Burkholderia cepacia), citrus nematode, and P. nicotianae, 
under different conditions of soil pH designed to result in different infection levels by the 
nematode. Soil pH of 4.5 reduced the infection rate of the nematode and also the fungus 
and reduced growth of seedlings, and amount of bacterial colonization. Bacteria CFU's 
(not identified to species) were recovered in significantly greater numbers in plants 



98 

nicotianae and T. semipenetrans at both pH levels. Root and stem fresh weights of 
fungus-infected plants treated with T. semipenetrans, alone or in combination with B. 
cepacia or B. megaterium were greater than for the plants treated with P. nicotianae only. 
Phytophthora nicotianae did not negatively affect seedling growth in the presence of 
either bacterium, whether alone or in combination with T. semipenetrans. However, 
neither bacterium suppressed the amount of P. nicotianae protein in citrus roots when 
used alone in a treatment. All treatments with Tylenchulus semipenetrans reduced the 
fungus protein in roots. These studies showed that nematode-induced changes in the 
rhizosphere community of bacteria may mitigate the virulence of P. nicotianae, perhaps 
by increasing plant tolerance, but not by inhibiting infection by the fungus. The results 
suggests that the "biological control" attributed to plant growth promoting rhizobacteria 
PGPR (Schroth and Han cock, 1981 ; Wei et al., 1991 ; Van Peer et al., 1991) may be less 
important than the effects these organisms have on the ability of plants to grow in the 
presence of pathogens. 

I then studied the possibility of direct inhibition of the fungus by the nematode in 
vitro bioassays were developed to determine the effects of eggs of two nematodes, T. 
semipenetrans and Meloidogyne arenaria, on P. nicotianae and Fusarium solani mycelial 
growth. Three treatments were used; live-surface sterilized eggs; heat-killed surface- 
sterilized eggs; and a water control. Live citrus nematode eggs suppressed mycelial 
growth of P. nicotianae and F. solani consistently, compared to heat-killed eggs and 
water controls. Root-knot nematode eggs had no comparable effect on mycelial growth 
of either fungus. The experiment demonstrated a species-specific, direct effect by the 
citrus nematode on the behavior of P. nicotianae and F. solani. 



99 

It is not surprising that T. semipenetrans may have adapted to protect its feeding 
site from a ubiquitous and sympatric pathogen of the citrus fibrous root cortex. In 
contrast to other nematode parasites of citrus, the citrus nematode has a narrow host 
range that includes citrus and only a few other woody perennials. It has the most refined 
and complex host-parasite relationship of any nematode parasite of citrus. The nematode 
is a good parasite, being relatively less virulent to citrus than other nematode parasites of 
the genus. These characteristics are consistent with the hypothesis that T. semipenetrans 
and its host (citrus) have co-evolved to a significant degree (Duncan, 1995). Indeed, 
selection pressure may favor a relatively benign response of the plant to infection by the 
nematode, because mechanisms that prevent infection of citrus roots by fungal pathogens 
would confer an adaptive advantage not only to the nematode, but to its host. 

This appears to be the first report of growth inhibition of a plant parasitic fungus 
by a plant parasitic nematode. These results suggest that, unlike many nematode fungus 
disease complexes, T. semipenetrans is unlikely to exacerbate plant damage caused by P. 
nicotianae in citrus. Indeed, in orchards infested with P. nicotianae, concomitant 
infection of citrus roots by T. semipenetrans may change the rhizoshpere chemistry and 
microbial community in ways that favor development of citrus roots. T. semipenetrans 
eggs inhibited two different classes of fungi, Oomycetes (P. nicotianae) and 
Hyphomycetes (F. solani), but had no negative effect on bacterial colonization of roots, 
thus exhibiting some selectivity toward fungi. Identification and characterization of the 
agent(s) in T. semipenetrans eggs that is responsible for P. nicotianae inhibition may 
provide a new class of fungicide for management of P. nicotianae. Identification of the 
chemical(s) may also facilitate identification of the nematode genes responsible for the 



100 

effect and may lead to a new means of introducing resistance against P. nicotianae into 
susceptible rootstocks. 





















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BIOGRAPHICAL SKETCH 
Fahiem E. El-Borai was born in El-Mansoura, Dakahleia governerate, Egypt on 6 

January, 1966. He received a B.S. in plant protection in 1987. Fahiem was appointed to 

a position as an assistant professor in the Plant Protection Department at El-Zagazig 

University in Egypt in 1988. He began working toward his Master of Science degree in 

the area of plant-parasitic nematodes. Fahiem conducted research and taught agricultural 

zoology courses (morphology, taxonomy and physiology) for undergraduate students at 

the Faculty of Agriculture, El-Zagazig University. 

Fahiem obtained his Master of Science degree in agricultural zoology 
(nematology) in 1993. He continued teaching at the same university until 1996. He was 
awarded a scholarship from the Egyptian government to pursue his Ph.D. studies abroad. 
In the summer of 1997, he enrolled in the graduate program (nematology) of the 
Entomology and Nematology Department at the University of Florida under Dr. Larry W. 
Duncan's supervision. He considers himself very fortunate to be enrolled in one of the 
greatest graduate programs in nematology, with excellent scientists who are a pleasure to 
work with. He completed his research at the Citrus Research and Education Center in 
Lake Alfred, Florida. 

After completing his Ph.D. program, Fahiem plans to work as a postdoctoral 
researcher to gain more experience in his subject area. Later, he will return to Egypt to 
fulfill an appointed position as an associate professor at El-Zagazig University. Fahiem 
will teach and conduct research and he hopes to deliver the excitement and enthusiasm to 
his students in Egypt that he has experienced from his professors in the U.S.A. 

119 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of PhU 




;arry w . Duncan, Chair 
Professor of Entomology and Nematology 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Jamels H. Graham 

bssor of Soil and Water Science 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Donald W. Dickson 

Professor of Entomology and Nematology 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 







Robert McSorley/ 

Professor of Entmnology and Nematology 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Z, yl^^v 



James L. Nation 

Professor of Entomology and Nematology 



This dissertation was submitted to the Graduate Faculty of the College of 
Agricultural and Life Sciences and to the Graduate School and was accepted as partial 
fulfillment of the requirements for the degree of Doctor of Philosophy. 

December 2001 





Dean, College of Agricultural antkLife 
Sciences 



Dean, Graduate School 





















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